Compositions and methods of use of targeting peptides against placenta and adipose tissues

ABSTRACT

The present invention concerns compositions comprising and methods of identification and use of targeting peptides for placenta or adipose tissue. In certain embodiments, the targeting peptides comprise part or all of SEQ ID NO:5-11, SEQ ID NO:13-22 or SEQ ID NO:144. The peptides may be attached to various therapeutic agents for targeted delivery. Adipose-targeting peptides may be used in methods for weight control, inducing weight loss and treating lipodystrophy syndrome. Adipose-targeting may also be accomplished using other binding moieties selectively targeted to adipose receptors, such as a prohibitin receptor protein complex. Placenta-targeting peptides may be used to interfere with pregnancy, induce labor and/or for targeted delivery of therapeutic agents to placenta and/or fetus. In other embodiments, receptors identified by binding to placenta-targeting peptides may be used to screen compounds for potential teratogenicity. An exemplary placental receptor is FcRn/β 2 M, and compounds that bind to FcRn/β 2 M are potential teratogens.

This application is a continuation in part of PCT applicationPCT/US01/27692, filed on Sep. 7, 2001, the entire text of which isincorporated herein by reference. This application claims priority fromU.S. Provisional Patent Application No. 60/231,266 filed Sep. 8, 2000,and U.S. patent application Ser. No. 09/765,101, filed Jan. 17, 2001.

This invention was made with U.S. government support under grants grantsCA90270, 1R1CA90810-01 and 1R01CA82976-01 from the National Institutesof Health. The U.S. government has certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention concerns the fields of molecular medicine andtargeted delivery of therapeutic agents. More specifically, the presentinvention relates to compositions and methods for identification and useof peptides that selectively target white adipose tissue and placenta invivo or in vitro. In other embodiments, the invention concernscompositions and methods for screening potential teratogenic agents.

2. Description of Related Art

Phage display is a technique in which a phage library expresses, forexample, a set of random peptide sequences of defined length,incorporated into a phage coat protein (e.g., Smith and Scott, Science228:1315-17, 1985; Smith and Scott, Meth. Enzymol. 21:228-57, 1993).Peptide sequences that bind to a target molecule, cell, tissue or organmay be identified by incubating a phage display library with the targetand selecting for bound peptides (biopanning) (e.g., Pasqualini andRuoslahti, Nature 380:364-66, 1996; Arap et al., Science 279:377-80,1998a). Unbound phage may be washed away and bound phage eluted andcollected. The collected phage may be amplified and taken throughfurther binding/amplification cycles to enrich the pool of peptides forthose that selectively and/or specifically bind to the target. With eachcycle, the proportion of phage in the pool that contain targetingpeptides for the target of interest is enriched. After several cycles,individual phage clones may be characterized by DNA sequencing toidentify the targeting peptide sequences.

Targeting peptides that exhibit selective and/or specific binding forplacenta or adipose tissues have not been previously reported in theliterature. Targeting peptides against placenta or adipose tissues wouldhave a variety of potential uses. Targeting peptides against adiposetissue could be used to control obesity and related conditions.Adipose-targeting peptides would also be of potential use to treat HIVrelated adipose malformations such as lipodystrophia and/orhyperlipidemia (see, e.g., Zhang et al., J. Clin. Endocrin. Metab.84:4274-77, 1999; Jain et al., Antiviral Res. 51:151-177, 2001; Raolinet al., Prog. Lipid Res. 41:27-65, 2002). Targeting peptides againstplacental tissue could be used to reduce harmful effects by tetragenicagents, to deliver therapeutic agents to the placenta and/or the fetusand to induce labor or spontaneous abortion. Placental receptorsidentified through the use of placental targeting peptides could be usedto screen for potential teratogens.

Presently available methods for control of weight include dieting andsurgical procedures. These often exhibit adverse effects and may notresult in long-term weight loss. Dieting includes both popular (fad)diets and the use of weight loss and appetite supplements. Fad diets areonly good for short-term weight loss and do not achieve long-term weightcontrol. They are often unhealthy, since many important nutrients aremissing from the diet. In addition, rapid weight loss can result indehydration. After losing weight, the dieters typically return to theiroriginal eating habits. This often results in weight gain that canexceed the subject's weight before dieting (yo yo effect).

Appetite suppressants such as Phentermen HCl, Meridia, Xernical,Adipex-P, Bontril and Ionomin may have adverse effects, such asaddiction, dry mouth, nausea, irritability, and constipation. Thesesupplements can also lead to more serious problems like eatingdisorders. Weight control through use of such supplements isineffective, with only limited weight loss achieved. Effective drugs forcontrolling weight, such as fenfluramine, were withdrawn from the marketdue to cardiotoxicity.

Surgical methods for weight reduction, such as liposuction and gastricbypass surgery, have many risks. Liposuction removes subcutaneous fatthrough a suction tube inserted into a small incision in the skin. Risksand complications may include scarring, bleeding, infection, change inskin sensation, pulmonary complications, skin loss, chronic pain, etc.In gastric bypass surgery, the patient has to go through the rest of hisor her life with a drastically altered stomach that can hold just two orthree ounces of food. Side effects may include nausea, diarrhea,bleeding, infection, bowel blockage caused by scar tissue, hernia andadverse reactions to general anesthesia. The most serious potential riskis leakage of fluid from the stomach or intestines, which may result inabdominal infection and the need for a second surgery. None of thepresently available methods for weight control is satisfactory and aneed exists for improved methods of weight loss and control.

Another adipose related disease state is lipodystrophy syndrome(s)related to HIV infection (e.g., Jain et al., Antiviral Res. 51:151-177,2001). Mortality rates from HIV infection have decreased substantiallyfollowing use of highly active antiretroviral therapy (HAART) (Id.)However, treatment with protease inhibitors as part of the HAARTprotocol appears to result in a number of lipid-related symptoms, suchas hyperlipidemia, fat redistribution with accumulation of abdominal andcervical fat, diabetes mellitus and insulin resistance (Jain et al.,2001; Yanovski et al., J. Clin. Endocrin. Metab. 84:1925-1931; Raulin etal., Prog. Lipid Res. 41:27-65, 2002). Although of minor significancecompared to the underlying HIV infection and possible development ofAIDS related complex (ARC) and/or AIDS, lipodystrophy syndrome adverselyaffects quality of life and may be associated with increased risk ofcoronary artery disease, heart attack, stroke and other adverse sideaffects of increased blood lipids. While treatment with metformin, aninsulin-sensitizing aget, has been reported to provide some alleviationof symptoms (Hadigan et al., J. Amer. Med. Assn. 284:472-477, 2000), aneed exists for more effective methods of treating HIV relatedlypodystrophy.

Teratogens fall into two classes. The first class includes compoundsthat are actively or passively transferred through the materno-fetalbarrier. Those target fetal development by altering cell-signalingpathways that control essential processes in the developing embryo, suchas angiogenesis (D'Amato, R. J., et al 1994. Proc. Natl. Acad. Sci. USA91: 40824085; Funnell, R. H. 1999. J. Allergy Clin. Immunol.103:337-342).

Teratogens of the second class interfere with fetal development byaffecting the delivery of nutrients to the embryo through the placenta(Maranghi, F., et al. 1998. Adv. Exp. Med. Biol. 444: 129-136; Rugh, R.1990. The Mouse: Its Reproduction and Development, Oxford SciencePublications, Oxford). Materno-fetal molecule exchange occurs byfiltration of blood from the maternal to the fetal side of the placentathrough several distinct cell layers. Teratogens that target theplacenta are thought to function by blocking receptors required fortransport of nutrients to the fetus (Beckman, D. A., et al. 1990.Teratology 41: 395-404). Present methods of treatment primarily involveavoiding exposure of the pregnant woman to teratogens. Such methods areineffective where the mother is unaware of her pregnancy, or for novelteratogens whose effect on fetal development have not yet beencharacterized. Because teratogens are identified by in vivo animaltesting, differences in placental receptors between humans and testanimals, such as mice, may result in the failure to identify teratogeniceffects until multiple birth defects are reported, such as in thethalidomide tragedy. A need exists for methods of identifying theplacental receptors for teratogens, in order to allow more accurateteratogen screening procedures.

SUMMARY OF THE INVENTION

The present invention solves a long-standing need in the art byproviding compositions and methods of preparation and use of targetingpeptides that are selective and/or specific for white adipose tissue orplacenta. In some embodiments, the invention concerns particulartargeting peptides selective or specific for adipose or placentaltissue, including but not limited to SEQ ID NO:5-11, SEQ ID NO:13-22 andSEQ ID NO:144. Other embodiments concern such targeting peptidesattached to therapeutic agents. In other embodiments, placental, adiposeor other targeting peptides may be used to selectively or specificallydeliver therapeutic agents to target tissues, such as white adiposetissue, placenta or fetal tissue. In certain embodiments, the subjectmethods concern the preparation and identification of targeting peptidesselective or specific for a given target cell, tissue or organ, such asadipose or placenta.

One embodiment of the invention concerns isolated peptides of 100 aminoacids or less in size, comprising at least 3 contiguous amino acids of atargeting peptide sequence, selected from any of SEQ ID NO:5-11, SEQ IDNO:13-22 and SEQ ID NO:144. In a preferred embodiment, the isolatedpeptide is 50 amino acids or less, more preferably 30 amino acids orless, more preferably 20 amino acids or less, more preferably 10 aminoacids or less, or even more preferably 5 amino acids or less in size. Inother preferred embodiments, the isolated peptide may comprise at least4, 5, 6, 7, 8 or 9 contiguous amino acids of a targeting peptidesequence, selected from any of SEQ ID NO:5-11, SEQ ID NO:13-22 and SEQID NO:144.

In certain embodiments, the isolated peptide may be attached to amolecule. In preferred embodiments, the attachment is a covalentattachment. In various embodiments, the molecule is a drug, achemotherapeutic agent, a radioisotope, a pro-apoptosis agent, ananti-angiogenic agent, a hormone, a cytokine, a growth factor, acytotoxic agent, a peptide, a protein, an antibiotic, an antibody, a Fabfragment of an antibody, a survival factor, an anti-apoptotic factor, ahormone antagonist, an imaging agent, a nucleic acid or an antigen.Those molecules are representative only and virtually any molecule maybe attached to a targeting peptide and/or administered to a subjectwithin the scope of the invention. In preferred embodiments, thepro-aptoptosis agent is gramicidin, magainin, mellitin, defensin,cecropin, (KLAKLAK)₂ (SEQ ID NO:1), (KLAKKLA)₂ (SEQ ID NO:2), (KAAKKAA)₂(SEQ ID NO:3) or (KLGKKLG)₃ (SEQ ID NO:4). In other preferredembodiments, the anti-angiogenic agent is angiostatin5, pigmentepithelium-derived factor, angiotensin, laminin peptides, fibronectinpeptides, plasminogen activator inhibitors, tissue metalloproteinaseinhibitors, interferons, interleukin 12, platelet factor 4, IP-10,Gro-β, thrombospondin, 2-methoxyoestradiol, proliferin-related protein,carboxiamidotriazole, CMIOI, Marimastat, pentosan polysulphate,angiopoietin 2 (Regeneron), interferon-alpha, herbimycin A, PNU145156E,16K prolactin fragment, Linomide, thalidomide, pentoxifylline,genistein, TNP-470, endostatin, paclitaxel, docetaxel, polyamines, aproteasome inhibitor, a kinase inhibitor, a signaling inhibitor (SU5416,SU6668, Sugen, South San Francisco, Calif.), accutin, cidofovir,vincristine, bleomycin, AGM-1470, platelet factor 4 or minocycline. Infurther preferred embodiments, the cytokine is interleukin 1 (IL-1),IL-2, IL-5, IL-10, IL-11, IL-12, IL-18, interferon-γ (IF-γ), IF-α, IF-β,tumor necrosis factor-α (TNF-α), or GM-CSF (granulocyte macrophagecolony stimulating factor). Such examples are representative only andare not intended to exclude other pro-apoptosis agents, anti-angiogenicagents or cytokines known in the art.

In various embodiments, targeting peptides attached to one or moretherapeutic agents may be administered to a subject, such as an animal,mammal, cat, dog, cow, pig, horse, sheep or human subject. Suchadministration may be of use for the treatment of various diseasestates. In certain embodiments, adipose-targeting peptides attached to acytocidal, pro-apoptotic, anti-angiogenic or other therapeutic agent maybe of use in methods to treat obesity, induce weight loss and/or totreat highly active antiretroviral therapy (HAART) associatedlipodystrophy syndrome. In other embodiments, placenta-targetingpeptides attached to such agents may be used, for example, to inducelabor or to terminate pregnancy.

In other embodiments of the invention, the isolated peptide may beattached to a macromolecular complex. In preferred embodiments, themacromolecular complex is a virus, a bacteriophage, a bacterium, aliposome, a microparticle, a magnetic bead, a yeast cell, a mammaliancell, a cell or a microdevice. These are representative examples onlyand macromolecular complexes within the scope of the present inventionmay include virtually any complex that may be attached to a targetingpeptide and administered to a subject. In other preferred embodiments,the isolated peptide may be attached to a eukaryotic expression vector,more preferably a gene therapy vector.

In another embodiment, the isolated peptide may be attached to a solidsupport, preferably magnetic beads, Sepharose beads, agarose beads, anitrocellulose membrane, a nylon membrane, a column chromatographymatrix, a high performance liquid chromatography (HPLC) matrix or a fastperformance liquid chromatography (FPLC) matrix.

Additional embodiments of the present invention concern fusion proteinscomprising at least 3 contiguous amino acids of a sequence selected fromany of SEQ ID NO:5-11, SEQ ID NO:13-22 and SEQ ID NO:144. In someembodiments, larger contiguous sequences, up to a full-length sequenceselected from any of SEQ ID NO:5-11, SEQ ID NO:13-22 and SEQ ID NO:144may be used.

Certain other embodiments concern compositions comprising the claimedisolated peptides or fusion proteins in a pharmaceutically acceptablecarrier. Further embodiments concern kits comprising the claimedisolated peptides or fusion proteins in one or more containers.

Other embodiments concern methods of targeted delivery comprisingselecting a targeting peptide for a desired organ, tissue or cell type,attaching said targeting peptide to a molecule, macromolecular complexor gene therapy vector, and providing said peptide attached to saidmolecule, complex or vector to a subject. Preferably, the targetingpeptide is selected to include at least 3 contiguous amino acids fromany of selected from any of SEQ ID NO:5-11, SEQ ID NO:13-22 and SEQ IDNO:144. In certain preferred embodiments, the organ, tissue or cell typeis white adipose or placenta. In other preferred embodiments, themolecule attached to the targeting peptide is a chemotherapeutic agent,an antigen or an imaging agent.

Other embodiments of the present invention concern isolated nucleicacids of 300 nucleotides or less in size, encoding a targeting peptide.In preferred embodiments, the isolated nucleic acid is 250, 225, 200,175, 150, 125, 100, 75, 50, 40, 30, 20 or even 10 nucleotides or less insize. In other preferred embodiments, the isolated nucleic acid isincorporated into a eukaryotic or a prokaryotic expression vector. Ineven more preferred embodiments, the vector is a plasmid, a cosmid, ayeast artificial chromosome (YAC), a bacterial artificial chromosome(BAC), a virus or a bacteriophage. In other preferred embodiments, theisolated nucleic acid is operatively linked to a leader sequence thatlocalizes the expressed peptide to the extracellular surface of a hostcell.

Additional embodiments of the present invention concern methods oftreating a disease state comprising selecting a targeting peptide thattargets cells associated with the disease state, attaching one or moremolecules effective to treat the disease state to the peptide, andadministering the peptide to a subject with the disease state.Preferably, the targeting peptide includes at least three contiguousamino acids selected from any of selected from any of SEQ ID NO:5-11,SEQ ID NO:13-22 and SEQ ID NO:144. In preferred embodiments the diseasestate is obesity, lipodystrophy or a related condition.

In certain embodiments, the methods concern Biopanning and RapidAnalysis of Selective Interactive Ligands (BRASIL), a novel method forphage display that results in decreased background of non-specific phagebinding, while retaining selective binding of phage to cell receptors.In preferred embodiments, targeting peptides are identified by exposinga subject to a phage display library, collecting samples of one or moreorgans, tissues or cell types, separating the samples into isolatedcells or small clumps of cells suspended in an aqueous phase, layeringthe aqueous phase over an organic phase, centrifuging the two phases sothat the cells are pelleted at the bottom of a centrifuge tube andcollecting phage from the pellet. In an even more preferred embodiment,the organic phase is dibutylphtalate.

In other embodiments, phage that bind to a target organ, tissue or celltype, for example to adipose tissue or placenta, may be pre-screened orpost-screened against a subject lacking that organ, tissue or cell type.Phage that bind to the subject lacking the target organ, tissue or celltype are removed from the library prior to screening in subjectspossessing the organ, tissue or cell type.

In preferred embodiments, targeting phage may be recovered from specificcell types or sub-types present in an organ or tissue after selection ofthe cell type by PALM (Positioning and Ablation with Laser Microbeams).PALM allows specific cell types to be selected from, for example, a thinsection of an organ or tissue. Phage may be recovered from the selectedsample.

In another embodiment, a phage display library displaying the antigenbinding portions of antibodies from a subject is prepared, the libraryis screened against one or more antigens and phage that bind to theantigens are collected. In more preferred embodiments, the antigen is atargeting peptide.

In certain embodiments, the methods and compositions may be used toidentify one or more receptors for a targeting peptide. In alternativeembodiments, the compositions and methods may be used to identifynaturally occurring ligands for known or newly identified receptors. Inpreferred embodiments, the receptor may be a placental receptor forteratogens. In some embodiments, the placental teratogen receptor(s)identified may be used for screening of potential teratogens forreceptor binding.

In some embodiments, the methods may comprise contacting a targetingpeptide to an organ, tissue or cell containing a receptor of interest,allowing the peptide to bind to the receptor, and identifying thereceptor by its binding to the peptide. In preferred embodiments, thetargeting peptide contains at least three contiguous amino acidsselected from any of selected from any of SEQ ID NO:5-11, SEQ IDNO:13-22 and SEQ ID NO:144. In other preferred embodiments, thetargeting peptide may comprise a portion of an antibody against thereceptor.

In alternative embodiments, the targeting peptide may contain a randomamino acid sequence. The skilled artisan will realize that thecontacting step can utilize intact organs, tissues or cells, or mayalternatively utilize homogenates or detergent extracts of the organs,tissues or cells. In certain embodiments, the cells to be contacted maybe genetically engineered to express a suspected receptor for thetargeting peptide. In a preferred embodiment, the targeting peptide ismodified with a reactive moiety that allows its covalent attachment tothe receptor. In a more preferred embodiment, the reactive moiety is aphotoreactive group that becomes covalently attached to the receptorwhen activated by light. In another preferred embodiment, the peptide isattached to a solid support and the receptor is purified by affinitychromatography. In other preferred embodiments, the solid supportcomprises magnetic beads, Sepharose beads, agarose beads, anitrocellulose membrane, a nylon membrane, a column chromatographymatrix, a high performance liquid chromatography (HPLC) matrix or a fastperformance liquid chromatography (FPLC) matrix.

In certain embodiments, the targeting peptide may inhibit the activityof a receptor upon binding to the receptor. The skilled artisan willrealize that receptor activity can be assayed by a variety of methodsknown in the art, including but not limited to catalytic activity andbinding activity. In other embodiments, binding of a targeting peptideto a receptor may inhibit a transport activity of the receptor.

In alternative embodiments, one or more ligands for a receptor ofinterest may be identified by the disclosed methods and compositions.One or more targeting peptides that mimic part or all of a naturallyoccurring ligand may be identified by phage display and biopanning invivo or in vitro. A naturally occurring ligand may be identified byhomology with a single targeting peptide that binds to the receptor, ora consensus motif of sequences that bind to the receptor. In otheralternative embodiments, an antibody may be prepared against one or moretargeting peptides that bind to a receptor of interest. Such antibodiesmay be used for identification or immunoaffinity purification of thenative ligand.

In certain embodiments, the targeting peptides of the present inventionare of use for the selective delivery of therapeutic agents, includingbut not limited to gene therapy vectors and fusion proteins, to specificorgans, tissues or cell types. The skilled artisan will realize that thescope of the claimed methods of use include any disease state that canbe treated by targeted delivery of a therapeutic agent to a desiredorgan, tissue or cell type. Although such disease states include thosewhere the diseased cells are confined to a specific organ, tissue orcell type, other disease states may be treated by an organ, tissue orcell type-targeting approach. In particular embodiments, the organ,tissue or cell type may comprise white adipose tissue or placenta.

Certain embodiments concern methods of obtaining antibodies against anantigen. In preferred embodiments, the antigen comprises one or moretargeting peptides. The targeting peptides are prepared and immobilizedon a solid support, serum-containing antibodies is added and antibodiesthat bind to the targeting peptides are collected.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. Validation of placenta homing phage. Phage bearing targetingpeptides were injected into pregnant mice and their recovery fromplacenta was compared to control fd-tet phage without targetingsequences. The placenta homing phage clones were: PA—TPKTSVT (SEQ IDNO:5), PC—RAPGGVR (SEQ ID NO:7), PE—LGLRSVG (SEQ ID NO:10), PF—YIRPFTL(SEQ ID NO:9).

FIG. 2. Validation of adipose homing peptides. Phage bearing targetingpeptides were injected into obese mice and their recovery from adiposetissue was compared to control fd-tet phage without targeting sequences.

FIG. 3. In vivo homing of the TPKTSVT (SEQ ID NO:5) motif to the mousevillous yolk sac (vys). (A) and (B) anti-phage immunohistochemistry; (C)and (D) anti-GST immunohistochemistry; (E) and (F) FITCimmunofluorescence in paraffin sections of placentas from 18 dpc (dayspost-conception) pregnant mice injected intravenously 6 h prior totissue processing with: (A) control insertless phage, (B) TPKTSVT (SEQID NO:5) phage, (C) control GST peptide, (D) TPKTSVT (SEQ ID NO:5)linked to GST peptide, (E) control FITC-peptide, or (F) TPKTSVT (SEQ IDNO:5) linked to FITC peptide. Homing of the TPKTSVT. (SEQ ID NO:5)peptide to the vys (dark arrows, D) and translocation to the embryoniccapillaries (light arrow, D) are indicated. Only the vys is shown in(C-F). Bar:100 μm (A-B); 20 μm (C-F).

FIG. 4. The TPKTSVT (SEQ ID NO:5) peptide specifically binds a placentaltransporter. (A) Recovery of indicated phage from embryos carried by 18dpc pregnant mice intravenously injected (tail vein) with 10¹⁰ TU of theindicated phage 6 h prior to phage recovery or immunohistochemistry.Control phage (light cross-hatch) showed no selective targeting ofplacenta. TPKTSVT (SEQ ID NO:5) phage were administered alone (darkcross-hatched) or co-administered with control-GST (black) or TPKTSVT(SEQ ID NO:5) linked to GST (white bar). Shown are mean+/−SEM (standarderror) from different embryos. (B-D) Anti-phage HRP immunohistochemistry(arrowheads) in paraffin sections of the vys from the correspondingmice, as indicated. Asterisks mark embryonic capillaries. Bar:20 μm.

FIG. 5. The TPKTSVT (SEQ ID NO:5) peptide blocks placental IgGtranscytosis. A 18 dpc pregnant mouse was intravenously injected with:(A) 100 mg of control GST fusion or (B) TPKTSVT (SEQ ID NO:5) linked toGST. The IgG distribution in the placenta after 6 h of peptidecirculation was detected by anti-mouse IgG immunohistochemistry inparaffin sections. Strong immunostaining is noted in the vys of miceinjected with a control GST fusion (arrowheads) and in the labyrinth ofwild-type mice injected with either TPKTSVT (SEQ ID NO:5) linked to GSTor control GST fusion (asterisks), but not in the vys of TPKTSVT (SEQ IDNO:5) linked to GST fusion. Bar: 100 μm. (C) and (D) A hypotheticalmodel for the TPKTSVT (SEQ ID NO:5) peptide function. (C) Normally, theFcRn/β₂m complex transports IgG from maternal circulation through thelabyrinth layer and then the yolk sac placenta (vys) and into theembryo. (D) Targeting of the FcRn/β₂m with TPKTSVT (SEQ ID NO:5) peptidecan block the receptor complex and the transport of IgG or phage-intothe embryo.

FIG. 6. Placental targeting by the TPKTSVT (SEQ ID NO:5) peptiderequires a functional FcRn/β₂m receptor complex. (A) Relative phagerecovery (placenta TU/ml to blood TU/ml ratio) from placentas derivedfrom 18 dpc pregnant wild-type or β₂m-deficient mice injected with 10¹⁰TU of control insertless phage (black) or TPKTSVT (SEQ ID NO:5) phage(white) 6 h prior to phage recovery or immunohistochemistry. Shown aremean+/− SEM from individual placentas. (B) and (C) Anti-phageimmunohistochemistry in paraffin sections of the placenta from 18 dpcpregnant β₂m-null mice intravenously injected with (B) TPKTSVT (SEQ IDNO:5) phage or (C) control placenta-homing phage displaying the YIRPFTL(SEQ ID NO:9) peptide 6 h prior to tissue processing. Staining of aplacenta-homing phage displaying: the unrelated peptide YIRPFTL (SEQ IDNO:9) is detected in the labyrinth blood vessels (arrowheads). Bar:100μm.

FIG. 7. TPKTSVT (SEQ ID NO:5) peptide inhibits mouse pregnancy and isteratogenic. (A) Pregnancy courses (representative from 5 independentexperiments) in mice subcutaneously injected with the indicated phage orpeptides were monitored by daily weighing of the mice upon eachinjection (arrows). (B) Appearance of a normally developed 20 dpc embryo(left) for control GST fusion treatment compared to a representative 20dpc embryo (right) resulting from TPKTSVT (SEQ ID NO:5) linked to GSTfusion treatment. A severely teratogenic phenotype is observed with theplacental targeting fusion peptide. (C) and (D) Hematoxylin/eosinstaining of 20 dpc paraffin-embedded placentas derived from miceinjected for 7 days with (C) control GST fusion or (D) the TPKTSVT (SEQID NO:5) linked to GST fusion peptide. Note hemorrhage (blackasterisks), necrosis (white asterisks), and fibrosis (arrowheads).Bar:500 μm (50 μm for insets).

FIG. 8. In vivo fat homing of the CKGGRAKDC (SEQ ID NO:22) motif ingenetically obese mice. (A) and (B) Anti-phage immunohistochemistry inparaffin sections of subcutaneous white fat from leptin-deficient miceintravenously injected 6 hr prior to tissue processing. (C) and (D)Peptide-FITC immunofluorescence in paraffin sections of subcutaneouswhite fat from leptin-deficient mice intravenously injected 6 hr priorto tissue processing. Mice were injected with (A) CKGGRAKDC (SEQ IDNO:22) phage, (B) control insertless phage, (C) CKGGRAKDC (SEQ ID NO:22)linked to FITC peptide, or (D) control CARAC (SEQ ID NO:12) linked toFITC peptide. Homing of the CKGGRAKDC (SEQ ID NO:22) peptide to fatblood vessels (arrows) and its uptake by fat endothelium are indicated.Bar: 10 μm.

FIG. 9. In vivo fat homing of the CKGGRAKDC (SEQ ID NO:22) motif inwild-type mice. (A), (C) and (E) Peptide-FITC immunofluorescence or (B),(D) and (F) lectin-rhodamine immunofluorescence in blood vessels of (A),(B), (E) and (F) subcutaneous white fat or (C) and (D) pancreas controlsdetected in paraffin-sectioned tissues from c57bl/6 mice intravenouslyco-injected 5 min prior to tissue processing. Mice were injected with(A), (B), (C) and (D) CKGGRAKDC (SEQ ID NO:22) linked to FITC peptideand lectin-rhodamine; or (E) and (F) control CARAC (SEQ ID NO:12) linkedto FITC peptide and lectin-rhodamine. (B), (D) and (F) Arrows showendothelium marked with lectin. (A) Arrows show homing of the CKGGRAKDC(SEQ ID NO:22) peptide to fat endothelium. Bar: 10 μm.

FIG. 10. Treatment of mouse obesity with fat vasculature-targetedapoptosis. Three cohorts (n=3) of (A) high-fat cafeteria diet-fed obesec57bl/6 mice; or (B) regular diet-fed old (˜1 year) c57bl/6 mice wereeach subcutaneously injected daily with equimolar amounts of theindicated peptides. Mouse body mass measurement was taken on days wheninjections were performed (injections were skipped on days for whichbody mass measurement is not shown). Error bars are SEM for themeasurements in three mice.

FIG. 11. Fat resorption induced by fat vasculature-targeted apoptosis.(A) Representative high-fat cafeteria diet-fed obese c57bl/6 mice; (B)and (C) representative regular diet-fed old (˜1 year) c57bl/6 mice; or(D) epididymal fat from representative regular diet-fed old c57bl/6 micefrom the experiment described in FIG. 10. Whole mice (A), subcutaneousfat (B), peritoneal fat (C) and total epididymal fat (D) from thecorresponding indicated treatments were photographed 1 week (A) or 3weeks (B), (C) and (D) after the beginning of subcutaneous injections.The injected peptides were CKGGRAKDC (SEQ ID NO:22) linked to (KLAKLAK)₂(SEQ ID NO:1) (left column), CARAC (SEQ ID NO:12) linked to (KLAKLAK)₂(SEQ ID NO:1) (middle column), and CKGGRAKDC (SEQ ID NO:22)co-administered with (KLAKLAK)₂ (SEQ ID NO:1) (right column).

FIG. 12. Destruction of fat blood vessels as a result of targetedapoptosis. (A) Tunnel immunohistochemistry, (B) secondary antibody onlynegative tunnel staining control and (C) and (D) hematoxylin/eosinstaining of white fat of mice. (A), (B) and (C) Mice were treated withCKGGRAKDC (SEQ ID NO:22) linked to (KLAKLAK)₂ (SEQ ID NO:1). (D) Micewere treated with CARAC (SEQ ID NO:12) linked to (KLAKLAK)₂ (SEQ IDNO:1). Apoptosis (arrows, (A)) and necrosis/lymphocyte infiltration(arrows, (C)) in response to CKGGRAKDC (SEQ ID NO:22) linked to(KLAKLAK)₂ (SEQ ID NO:1) treatment are indicated. Bar: 10 μm.

FIG. 13. Spleen targeting in vitro using BRASIL. Binding of Fab clones#2, #6, #10, #12 and control Fab clone NPC-3TT were directly compared toeach other.

FIG. 14. Spleen targeting in vivo using BRASIL. Binding of Fab clones#2, #6, #10 and #12 to spleen tissue was compared to binding of Fabcontrol clone NPC-3TT.

FIG. 15. Spleen targeting in vivo using BRASIL. Binding of Fab clones#2, #6, #10, #12 was compared to binding of Fd-tet phage.

FIG. 16. Spleen targeting in vivo using BRASIL. Binding of Fab clone #10to spleen tissue was compared to binding of Fab control clone NPC-3TTand Fd-tet phage.

FIG. 17. Binding of Fab clone #10 to spleen versus bone marrow incomparison to Fd-tet phage.

FIG. 18. Binding of β3 cytoplasmic domain-selected phage to immobilizedproteins. GST fusion proteins or GST alone were coated on microtiterwells at 10 μg/ml and used to bind phage-expressing endostatin targetingpeptides. Each phage is identified by the peptide sequence it displayed:GILDTYRGSP (SEQ ID NO:30); YDWWYPWSW (SEQ ID NO:29); CLRQSYSYNC (SEQ IDNO:38); SDNRYIGSW (SEQ ID NO:31); CEQRQTQEGC (SEQ ID NO:27); CFQNRC (SEQID NO:36). The data represent the mean colony counts from triplicatewells, with standard error of less than 10% of the mean.

FIG. 19. Binding of β5 cytoplasmic domain-selected phage to immobilizedproteins. GST fusion proteins or GST alone were coated on microtiterwells at 10 μg/ml and used to bind phage-expressing endostatin bindingpeptides. Each phage is identified by the peptide sequence it displayed:(A) DEEGYYMMR (SEQ ID NO:44); (B) KQFSYRYIL (SEQ ID NO:45); (C)CEPYWDGWFC (SEQ ID NO:40); (D) VVISYSMPD (SEQ ID NO:46); and (E)CYIWPDSGLC (SEQ ID NO:39). The data represent the mean colony countsfrom triplicate wells, with standard error less than 10% of the mean.

FIG. 20. Binding of the cytoplasmic-domain binding phage to β3immobilized protein and inhibition with the synthetic peptide. Phagewere incubated on wells coated with GST-β3cyto in the presence ofincreasing concentrations of the corresponding synthetic peptide or acontrol peptide. The data represent the mean colony counts fromtriplicate wells, with standard error less than 10% of the mean.

FIG. 21. Binding of the cytoplasmic-domain binding phage to 35immobilized protein and inhibition with the synthetic peptide. Phagewere incubated on wells coated with GST-β5cyto in the presence ofincreasing concentrations of the corresponding synthetic peptide or acontrol peptide. The data represent the mean colony counts fromtriplicate wells, with standard error less than 10% of the mean.

FIG. 22. Binding of phage to immobilized β3-GST and β5-GST afterphosphorylation. Phage were phosphorylated with Fyn kinase. Insertlessphage were used as a control. Phage were incubated on wells coated withGST-β3cyto or GST-β3cyto. The data represent the mean colony counts fromtriplicate wells, with standard error less than 10% of the mean.

FIG. 23. Binding of phage to immobilized GST fusion proteins afterphosphorylation. Phages were phosphorylated with Fyn kinase. Insertlessphage was used as a control. Phage were incubated on wells coated withGST-cytoplasmic domains. The data represent the mean of colony countsfrom triplicate wells, with standard error less than 10% of the mean.

FIG. 24. Effect of integrin cytoplasmic domain binding peptides on cellproliferation. Serum-deprived cells were cultured for 24 h. and theproliferation was determined by [³H] thymidine (1 μCi/ml) uptakemeasurements. In a positive control, VEGF was added back toserum-starved cells. Each experiment was performed three times withtriplicates, and the results were expressed as the mean+/SD.

FIG. 25. Effect of penetratin peptide chimeras on endothelial cellmigration. Cell migration assay were performed in a 48-wellmicrochemotaxis chamber. Five random high-power fields (magnitude 40×)were counted in each well. The results show that both β3-integrincytoplasmic domain-binding peptides (Y-18 and TYR-11) increase cellmigration while penetratin does not affect the cells.

FIG. 26. Penetratin peptide chimera binding to the β5 cytoplasmic domaininduces programmed cell death. 10⁶ HUVEC cells were harvested incomplete media and 15 μM penetratin peptide chimeras were added to thecells. After four, eight and twelve hours the cells were stained withPropidium Iodide (PI) and induction of apoptosis was analyzed bycytometric analysis. (a) Profile obtained with starved cells after 24 h.(b) Confluent cells in complete media. (c) 15 μM of penetratin afterfour hours. (d) 15 μM of VISY-penetratin chimera after four hours. Cellsanalyzed after eight and twelve hours showed similar profiles for thepercentage of G₀/G₁.

FIG. 27. Specificity of the antibodies raised against β3- or β5-selectedphage (ELISA). Increasing dilutions of sera obtained after threeimmunizations with GLDTYRGSP. (SEQ ID NO:30) or SDNRYIGSW (SEQ ID NO:31)conjugated to KLH were incubated on microtiter wells coated with 10 μgof SDNRYIGSW (SEQ ID NO:31, Y-18), GIDTYRGSP (SEQ ID NO:30, TYR-11) orcontrol peptides. Preimmune sera were used as controls. After incubationwith HRP-goat anti-rabbit, OD was measured at 405 mm. The data representthe means from triplicate wells, with standard error less than 10%.

FIG. 28. Specificity of the antibodies raised against β3- or β5-selectedphage (ELISA). Sera obtained after three immunizations with SDNRYIGSW(SEQ ID NO:31, Y-18) or GLDTYRGSP (SEQ ID NO:30, TYR-11) conjugated toKLH were incubated in microtiter wells coated with 10 μg of TYR-11 orY-18. GLDTYRGSP (SEQ ID NO:30) or SDNRYIGSW (SEQ ID NO:31) and controlpeptides were added in solution. After incubation with HRP goatanti-rabbit, OD was measured at 405 nm. The data represent the meansfrom triplicate wells, with standard error less than 10%. Peptides addedin solution specifically block the reactivity with the immobilizedpeptides.

FIG. 29A. Competitive binding of Annexin V to β5 integrin with VISYpeptide. Binding assays were performed by ELISA.

FIG. 29B. Relative levels of binding of anti-Annexin V antibody topurified Annexin V protein and VISY peptide.

FIG. 30. Chimeric peptide containing VISY peptide linked to penetratin(antennapedia) induces apoptosis. VISY induced apoptosis was inhibitedby addition of a caspase inhibitor (zVAD).

FIG. 31. APA-binding phage specifically bind tumors. Equal amounts ofphage were injected into the tail veins of mice bearingMDA-MB435-derived tumors and phage were recovered after perfusion. Meanvalues for phage recovered from the tumor or control tissue (brain) andthe standard error from triplicate platings are shown.

FIG. 32. CPRECESIC (SEQ ID NO:56) is a specific inhibitor of APAactivity. APA enzyme activity was assayed in the presence of increasingconcentrations of either GACVRLSACGA (SEQ ID NO:57) (control) orCPRECESIC (SEQ ID NO:56) peptide. The IC₅₀ for APA inhibition byCPRECESIC (SEQ ID NO:56) was estimated at 800 μM. Error bars are thestandard error of the means of triplicate wells. The experiment wasrepeated three times with similar results.

FIG. 33. CPRECESIC (SEQ ID NO:56) inhibits HUVEC migration. HUVECs werestimulated with VEGF-A (10 ng/ml). The assay was performed in a Boydenmicrochemotaxis chamber, and cells were allowed to migrate through an8-μm pore filter for 5 h at 37° C. GACVRLSACGA (SEQ ID NO:57) (control)and CPRECESIC (SEQ ID NO:56) peptides were tested at 1 mM concentration.Migrated cells were stained and five high-power fields (magnitude 100×)for each microwell were counted. Error bars are the standard error ofthe means of triplicate microwells.

FIG. 34. CPRECESIC (SEQ ID NO:56) inhibits HUVEC proliferation. Cellswere stimulated with VEGF-A (10 ng/ml), and growth was evaluated at theindicated times by a colorimetric assay based on crystal violetstaining. Error bars are the standard error of the means of triplicatewells. Each experiment was repeated at least twice with similar results.

FIG. 35. Protocol for in vivo biopanning for phage targeted in mousepancreas, kidneys, liver, lungs and adrenal gland.

FIG. 36. Protocol for recovery of phage by infection of E. coli orrecovery of phage DNA by amplification and subcloning.

FIG. 37. Pancreatic islet targeting peptides and homologous proteins.Candidate endogenous proteins mimicked by the pancreatic islet targetingpeptides CVSNPRWKC (SEQ ID NO:131), CVPRRWDVC (SEQ ID NO:128), CQHTSGRGC(SEQ ID NO:129) and CRARGWLLC (SEQ ID NO:130), identified by standardhomology searches.

FIG. 38. Pancreatic islet targeting peptides and homologous proteins.Candidate endogenous proteins mimicked by the pancreatic islet targetingpeptides CGGVHALRC (SEQ ID NO:98), CFNRTWIGC (SEQ ID NO:132) andCWSRQGGC (SEQ ID NO:134, identified by standard homology searches.

FIG. 39. Pancreatic islet targeting peptides and homologous proteins.Candidate endogenous proteins mimicked by the pancreatic islet targetingpeptides CLASGMDAC (SEQ ID NO:138), CHDERTGRC (SEQ ID NO:139), CAHHALMEC(SEQ ID NO:140) and CMQGARTSC (SEQ ID NO:142), identified by standardhomology searches.

FIG. 40. Pancreatic islet targeting peptides and homologous proteins.Candidate endogenous proteins mimicked by the pancreatic islet targetingpeptides CHVLWSTRC (SEQ ID NO:135), CMSSPGVAC (SEQ ID NO:137) andCLGLLMAGC (SEQ ID NO:136), identified by standard homology searches.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

As used herein in the specification, “a” or “an”, may mean one or more.As used herein in the claim(s), in conjunction with the word“comprising,” the words “a” or “an”, may mean one or more than one. Asused herein “another” may mean at least a second or more of an item.

A “targeting peptide” is a peptide comprising a contiguous sequence ofamino acids, which is characterized by selective localization to anorgan, tissue or cell type. Selective localization may be determined,for example, by methods disclosed below, wherein the putative targetingpeptide sequence is incorporated into a protein that is displayed on theouter surface of a phage. Administration to a subject of a library ofsuch phage that have been genetically engineered to express a multitudeof such targeting peptides of different amino acid sequence is followedby collection of one or more organs, tissues or cell types from thesubject and identification of phage found in that organ, tissue or celltype. A phage expressing a targeting peptide sequence is considered tobe selectively localized to a tissue or organ if it exhibits greaterbinding in that tissue or organ compared to a control tissue or organ.Preferably, selective localization of a targeting peptide should resultin a two-fold or higher enrichment of the phage in the target organ,tissue or cell type, compared to a control organ, tissue or cell type.Selective localization resulting in at least a three-fold, four-fold,five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold orhigher enrichment in the target organ compared to a control organ,tissue or cell type is more preferred. Alternatively, a phage expressinga targeting peptide sequence that exhibits selective localizationpreferably shows an increased enrichment in the target organ compared toa control organ when phage recovered from the target organ arereinjected into a second host for another round of screening. Furtherenrichment may be exhibited following a third round of screening.Another alternative means to determine selective localization is thatphage expressing the putative target peptide preferably exhibit atwo-fold, more preferably a three-fold or higher enrichment in thetarget organ compared to control phage that express a non-specificpeptide or that have not been genetically engineered to express anyputative target peptides. Another means to determine selectivelocalization is that localization to the target organ of phageexpressing the target peptide is at least partially blocked by theco-administration of a synthetic peptide containing the target peptidesequence. “Targeting peptide” and “homing peptide” are used synonymouslyherein.

A “phage display library” means a collection of phage that have beengenetically engineered to express a set of putative targeting peptideson their outer surface. In preferred embodiments, DNA sequences encodingthe putative targeting peptides are inserted in frame into a geneencoding a phage capsule protein. In other preferred embodiments, theputative targeting peptide sequences are in part random mixtures of alltwenty amino acids and in part non-random. In certain preferredembodiments the putative targeting peptides of the phage display libraryexhibit one or more cysteine residues at fixed locations within thetargeting peptide sequence. Cysteines may be used, for example, tocreate a cyclic peptide.

A “macromolecular complex” refers to a collection of molecules that maybe random, ordered or partially ordered in their arrangement. The termencompasses biological organisms such as bacteriophage, viruses,bacteria, unicellular pathogenic organisms, multicellular pathogenicorganisms and prokaryotic or eukaryotic cells. The term also encompassesnon-living assemblages of molecules, such as liposomes, microcapsules,microparticles, magnetic beads and microdevices. The only requirement isthat the complex contains more than one molecule. The molecules may beidentical, or may differ from each other.

A “receptor” for a targeting peptide includes but is not limited to anymolecule or macromolecular complex that binds to a targeting peptide.Non-limiting examples of receptors include peptides, proteins,glycoproteins, lipoproteins, epitopes, lipids, carbohydrates,multi-molecular structures, a specific conformation of one or moremolecules and a morphoanatomic entity. In preferred embodiments, a“receptor” is a naturally occurring molecule or complex of moleculesthat is present on the lumenal surface of cells forming blood vesselswithin a target organ, tissue or cell type.

A “subject” refers generally to a mammal. In certain preferredembodiments, the subject is a mouse or rabbit. In even more preferredembodiments, the subject is a human.

Phage Display

Recently, an in vivo selection system was developed using phage displaylibraries to identify organ, tissue or cell type-targeting peptides in amouse model system. Phage display libraries expressing transgenicpeptides on the surface of bacteriophage were initially developed to mapepitope binding sites of immunoglobulins (Smith, G P and Scott, J K,1985. Science, 228:1315-1317, Smith, G P and Scott, J K, 1993. Meth.Enzymol. 21:228-257). Such libraries can be generated by insertingrandom oligonucleotides into cDNAs encoding a phage surface protein,generating collections of phage particles displaying unique peptides inas many as 10⁹ permutations. (Pasqualini, R. and Ruoslahti, E. 1996,Nature, 380: 364-366; Arap et al, 1998a; Arap et al., 1998b, Curr. Opin.Oncol. 10:560-565).

Intravenous administration of phage display libraries to mice wasfollowed by the recovery of phage from individual organs (Pasqualini andRuoslahti, 1996). Phage were recovered that were capable of selectivehoming to the vascular beds of different mouse organs, tissues or celltypes, based on the specific targeting peptide sequences expressed onthe outer surface of the phage (Pasqualini and Ruoslahti, 1996). Avariety of organ and tumor-homing peptides have been identified by thismethod (Rajotte et al., 1998, J. Clin. Invest. 102:430-437; Rajotte etal, 1999, J. Biol. Chem. 274:11593-11598; Koivunen et al., 1999a, NatureBiotechnol. 17: 768-774; Burg M, et al., 1999, Cancer Res. 58:2869-2874;Pasqualini, 1999, Quart. J. Nucl. Med. 43:159-162). Each of thosetargeting peptides bound to different receptors that were selectivelyexpressed on the vasculature of the mouse target tissue (Pasqualini,1999; Pasqualini et al., 2000; Folkman J. Nature Biotechnol. 15:510,1997; Folkman J. Nature Med 1:27-31, 1995). Tumor-homing peptides boundto receptors that were upregulated in the tumor angiogenic vasculatureof mice (Brooks, P. C., et al. Cell 79:1157-1164, 1994b; Pasqualini etal., 2000). In addition to identifying individual targeting peptidesselective for an organ, tissue or cell type (Pasqualini and Ruoslahti,1996; Arap et al, 1998a; Koivunen et al., Methods Mol. Biol. 129: 3-17,1999b), this system has been used to identify endothelial cell surfacemarkers that are expressed in mice in vivo (Rajotte and Ruoslahti,1999).

Attachment of therapeutic agents to targeting peptides resulted in theselective delivery of the agent to a desired organ, tissue or cell typein the mouse model system. Targeted delivery of chemotherapeutic agentsand proapoptotic peptides to receptors located in tumor angiogenicvasculature resulted in a marked increase in therapeutic efficacy and adecrease in systemic toxicity in tumor bearing mouse models (Arap etal., 1998a, 1998b; Ellerby et al., Nature Med 9:1032-1038, 1999).

The methods described herein for identification of targeting peptidesinvolve the in vivo administration of phage display libraries. Variousmethods of phage display and methods for producing diverse populationsof peptides are well known in the art. For example, U.S. Pat. Nos.5,223,409; 5,622,699 and 6,068,829 disclose methods for preparing aphage library. The phage display technique involves geneticallymanipulating bacteriophage so that small peptides can be expressed ontheir surface (Smith and Scott, 1985, 1993). The potential range ofapplications for this technique is quite broad, and the past decade hasseen considerable progress in the construction of phage-displayedpeptide libraries and in the development of screening methods in whichthe libraries are used to isolate peptide ligands. For example, the useof peptide libraries has made it possible to characterize interactingsites and receptor-ligand binding motifs within many proteins, such asantibodies involved in inflammatory reactions or integrins that mediatecellular adherence. This method has also been used to identify novelpeptide ligands that serve as leads to the development of peptidomimeticdrugs or imaging agents (Arap et al., 1998a). In addition to peptides,larger protein domains such as single-chain antibodies can also bedisplayed on the surface of phage particles (Arap et al., 1998a).

Targeting peptides selective for a given organ, tissue or cell type canbe isolated by “biopanning” (Pasqualini and Ruoslahti, 1996; Pasqualini,1999). In brief, a library of phage containing putative targetingpeptides is administered to an animal or human and samples of organs,tissues or cell types containing phage are collected. In preferredembodiments utilizing filamentous phage, the phage may be propagated invitro between rounds of biopanning in pilus-positive bacteria. Thebacteria are not lysed by the phage but rather secrete multiple copiesof phage that display a particular insert. Phage that bind to a targetmolecule can be eluted from the target organ, tissue or cell type andthen amplified by growing them in host bacteria. If desired, theamplified phage can be administered to a host and samples of organs,tissues or cell types again collected. Multiple rounds of biopanning canbe performed until a population of selective binders is obtained. Theamino acid sequence of the peptides is determined by sequencing the DNAcorresponding to the targeting peptide insert in the phage genome. Theidentified targeting peptide can then be produced as a synthetic peptideby standard protein chemistry techniques (Arap et al., 1998a, Smith andScott, 1985). This approach allows circulating targeting peptides to bedetected in an unbiased functional assay, without any preconceivednotions about the nature of their target. Once a candidate target isidentified as the receptor of a targeting peptide, it can be isolated,purified and cloned by using standard biochemical methods (Pasqualini,1999; Rajotte and Ruoslahti, 1999).

In certain embodiments, a subtraction protocol is used with to furtherreduce background phage binding. The purpose of subtraction is to removephage from the library that bind to cells other than the cell ofinterest, or that bind to inactivated cells. In alternative embodiments,the phage library may be prescreened against a subject who does notpossess the targeted cell, tissue or organ. For example,placenta-binding peptides may be identified after prescreening a libraryagainst a male or non-pregnant female subject After subtraction thelibrary may be screened against the cell, tissue or organ of interest.In another alternative embodiment, an unstimulated, quiescent cell type,tissue or organ may be screened against the library and binding phageremoved. The cell line, tissue or organ is then activated, for exampleby administration of a hormone, growth factor, cytokine or chemokine andthe activated cell type, tissue or organ screened against the subtractedphage library.

Other methods of subtraction protocols are known and may be used in thepractice of the present invention, for example as disclosed in U.S. Pat.Nos. 5,840,841, 5,705,610, 5,670,312 and 5,492,807.

Choice of Phage Display System.

Previous in vivo selection studies performed in mice preferentiallyemployed libraries of random peptides expressed as fusion proteins withthe gene III capsule protein in the fUSE5 vector (Pasqualini andRuoslahti, 1996). The number and diversity of individual clones presentin a given library is a significant factor for the success of in vivoselection. It is preferred to use primary libraries, which are lesslikely to have an over-representation of defective phage clones(Koivunen et al., 1999b). The preparation of a library should beoptimized to between 10⁸-10⁹ transducing units (T.U.)/ml. In certainembodiments, a bulk amplification strategy is applied between each roundof selection.

Phage libraries displaying linear, cyclic, or double cyclic peptides maybe used within the scope of the present invention. However, phagelibraries displaying 3 to 10 random residues in a cyclic insert(CX₃₋₁₀C) are preferred, since single cyclic peptides tend to have ahigher affinity for the target organ than linear peptides. Librariesdisplaying double-cyclic peptides (such as CX₃C X₃CX₃C; Rojotte et al.,1998) have been successfully used. However, the production of thecognate synthetic peptides, although possible, can be complex due to themultiple conformers with different disulfide bridge arrangements.

Identification of Homing Peptides and Receptors by In Vivo Phage Displayin Mice.

In vivo selection of peptides from phage-display peptide librariesadministered to mice has been used to identify targeting peptidesselective for normal mouse brain, kidney, lung, skin, pancreas, retina,intestine, uterus, prostate, and adrenal gland (Pasqualini andRuoslahti, 1996; Pasqualini, 1999; Rajotte et al., 1998). These resultsshow that the vascular endothelium of normal organs is sufficientlyheterogeneous to allow differential targeting with peptide probes(Pasqualini and Ruoslahti, 1996; Rajotte et al., 1998). A means ofidentifying peptides that home to the angiogenic vasculature of tumorshas been devised, as described below. A panel of peptide motifs thattarget the blood vessels of tumor xenografts in nude mice has beenassembled (Arap et al., 1998a; reviewed in Pasqualini, 1999). Thesemotifs include the sequences RGD-4C, NGR, and GSL. The RGD-4C peptidehas previously been identified as selectively binding av integrins andhas been shown to home to the vasculature of tumor xenografts in nudemice (Arap et al., 1998a, 1998b; Pasqualini et al., Nature Biotechnol15: 542-546, 1997).

The receptors for the tumor homing RGD4C targeting peptide has beenidentified as αv integrins (Pasqualini et al., 1997). The αv integrinsplay an important role in angiogenesis. The αvβ3 and αvβ5 integrins areabsent or expressed at low levels in normal endothelial cells but areinduced in angiogenic vasculature of tumors (Brooks P C, Clark R A,Cheresh D A. Science, 264: 569-571, 1994, 1994; Hammes H P, Brownlee M,Jonczyk A, Sutter A, and Preissner K T. Nature Med. 2: 529-533, 1996.).Aminopeptidase N/CD13 has recently been identified as an angiogenicreceptor for the NGR motif (Burg, M. A., et al. Cancer Res. 59,2869-2874, 1999.). Aminopeptidase N/CD13 is strongly expressed not onlyin the angiogenic blood vessels of prostate cancer in TRAMP mice butalso in the normal epithelial prostate tissue.

Tumor-homing phage co-localize with their receptors in the angiogenicvasculature of tumors but not in non-angiogenic blood vessels in normaltissues (Arap et al., 1998b). Immunohistochemical evidence shows thatvascular targeting phage bind to human tumor blood vessels in tissuesections (Pasqualini et al., 2000) but not to normal blood vessels. Anegative control phage with no insert (fd phage) did not bind to normalor tumor tissue sections. The expression of the angiogenic receptors wasevaluated in cell lines, in non-proliferating blood vessels and inactivated blood vessels of tumors and other angiogenic tissues such ascorpus luteum. Flow cytometry and immunohistochemistry showed that thesereceptors are expressed, in a number of tumor cells and in activatedHUVECs (data not shown). The angiogenic receptors were not detected inthe vasculature of normal organs of mouse or human tissues.

The distribution of these receptors was analyzed by immunohistochemistryin tumor cells, tumor vasculature, and normal vasculature. Alpha vintegrins, CD13, aminopeptidase A, NG2, and MMP-2/MMP-9—the knownreceptors in tumor blood vessels—are specifically expressed inangiogenic endothelial cells and pericytes of both human and murineorigin. Angiogenic neovasculature expresses markers that are eitherexpressed at very low levels or not at all in non-proliferatingendothelial cells (not shown).

The markers of angiogenic endothelium include receptors for vasculargrowth factors, such as specific subtypes of VEGF and basic FGFreceptors, and αv integrins, among many others (Mustonen T and AlitaloK. J. Cell Biol. 129:895-898, 1995.). Thus far, identification andisolation of novel molecules characteristic of angiogenic vasculaturehas been slow, mainly because endothelial cells undergo dramaticphenotypic changes when grown in culture (Watson et al., Science,268:447-448, 1995).

Many of these tumor vascular markers are proteases and some of themarkers also serve as viral receptors. Alpha v integrins are receptorsfor adenoviruses (Wickham et al., Cancer Immunol. Immunother.45:149-151, 1997c) and CD13 is a receptor for coronaviruses (Look et al.N. J. Clin. Invest. 83:1299-1307, 1989.). MMP-2 and MMP-9 are receptorsfor echoviruses (Koivunen et al., 1999a). Aminopeptidase A also appearsto be a viral receptor. Bacteriophage may use the same cellularreceptors as eukaryotic viruses. These findings suggest that receptorsisolated by in vivo phage display will have cell internalizationcapability, a key feature for utilizing the identified peptide motifs astargeted gene therapy carriers.

Targeted Delivery

Peptides that home to tumor vasculature have been coupled to cytotoxicdrugs or proapoptotic peptides to yield compounds that were moreeffective and less toxic than the parental compounds in experimentalmodels of mice bearing tumor xenografts (Arap et al., 1998a; Ellerby etal, 1999). The insertion of the RGD-4C peptide into a surface protein ofan adenovirus has produced an adenoviral vector that may be used fortumor targeted gene therapy (Arap et al., 1998b).

BRASIL

In preferred embodiments, separation of phage bound to the cells of atarget organ, tissue or cell type from unbound phage is achieved usingthe BRASIL technique (PCT Patent Application PCT/US01/28124 entitled,“Biopanning and Rapid Analysis of Selective Interactive Ligands(BRASIL)” by Arap et al., filed Sep. 7, 2001, incorporated herein byreference in its entirety). In BRASIL (Biopanning and Rapid Analysis ofSoluble Interactive Ligands), an organ, tissue or cell type is gentlyseparated into cells or small clumps of cells that are suspended in anaqueous phase. The aqueous phase is layered over an organic phase ofappropriate density and centrifuged. Cells attached to bound phage arepelleted at the bottom of the centrifuge tube, while unbound phageremain in the aqueous phase. This allows a more efficient separation ofbound from unbound phage, while maintaining the binding interactionbetween phage and cell. BRASIL may be performed in an in vivo protocol,in which organs, tissues or cell types are exposed to a phage displaylibrary by intravenous administration, or by an ex vivo protocol, wherethe cells are exposed to the phage library in the aqueous phase beforecentrifugation.

Preparation of Large Scale Primary Libraries

In certain embodiments, primary phage libraries are amplified beforeinjection into a human subject. A phage library is prepared by ligatingtargeting peptide-encoding sequences into a phage vector, such as fUSE5.The vector is transformed into pilus negative host E. coli such asstrain MC1061. The bacteria are grown overnight and then aliquots arefrozen to provide stock for library production. Use of pilus negativebacteria avoids the bias in libraries that arises from differentialinfection of pilus positive bacteria by different targeting peptidesequences.

To freeze, bacteria are pelleted from two thirds of a primary libraryculture (5 liters) at 4000×g for 10 min. Bacteria are resuspended andwashed twice with 500 ml of 10% glycerol in water, then frozen in anethanol/dry ice bath and stored at −80° C.

For amplification, 1.5 ml of frozen bacteria are inoculated into 5liters of LB medium with 20 μg/ml tetracycline and grown overnight.Thirty minutes after inoculation, a serial dilution is plated on LB/tetplates to verify the viability of the culture. If the number of viablebacteria is less than 5-10 times the number of individual clones in thelibrary (1-2×10⁸) the culture is discarded.

After growing the bacterial culture overnight, phage are precipitated.About ¼ to ⅓ of the bacterial culture is kept growing overnight in 5liters of fresh medium and the cycle is repeated up to 5 times. Phageare pooled from all cycles and used for injection into human subjects.

Human Subjects

The methods used for phage display biopanning in the mouse model systemrequire substantial improvements for use with humans. Techniques forbiopanning in human subjects are disclosed in PCT Patent ApplicationPCT/US01/28044, filed Sep. 7, 2001, the entire text of which isincorporated herein by reference. In general, humans suitable for usewith phage display are either brain dead or terminal wean patients. Theamount of phage library (preferably primary library) required foradministration must be significantly increased, preferably to 10¹⁴ TU orhigher, preferably administered intravenously in approximately 200 ml ofRinger lactate solution over about a 10 minute period.

The amount of phage required for use in humans has required substantialimprovement of the mouse protocol, increasing the amount of phageavailable for injection by five orders of magnitude. To produce suchlarge phage libraries, the transformed bacterial pellets recovered fromup to 500 to 1000 transformations are amplified up to 10 times in thebacterial host, recovering the phage from each round of amplificationand adding LB Tet medium to the bacterial pellet for collection ofadditional phage. The phage inserts remain stable under these conditionsand phage may be pooled to form the large phage display library requiredfor humans.

Samples of various organs and tissues are collected startingapproximately 15 minutes after injection of the phage library. Samplesare processed as described below and phage collected from each organ,tissue or cell type of interest for DNA sequencing to determine theamino acid sequences of targeting peptides.

With humans, the opportunities for enrichment by multiple rounds ofbiopanning are severely restricted, compared to the mouse model system.A substantial improvement in the biopanning technique involves polyorgantargeting.

Polyorgan Targeting

In the standard protocol for phage display biopanning, phage from asingle organ are collected, amplified and injected into a new host,where tissue from the same organ is collected for phage rescue and a newround of biopanning. This protocol is feasible in animal subjects.However, the limited availability and expense of processing samples fromhumans requires an improvement in the protocol.

It is possible to pool phage collected from multiple organs after afirst round of biopanning and inject the pooled sample into a newsubject, where each of the multiple organs may be collected again forphage rescue. The polyorgan targeting protocol may be repeated for asmany rounds of biopanning as desired. In this manner, it is possible tosignificantly reduce the number of subjects required for isolation oftargeting peptides for multiple organs, while still achievingsubstantial enrichment of the organ-homing phage.

In preferred embodiments, phage are recovered from human organs, tissuesor cell types after injection of a phage display library into a humansubject. In certain embodiments, phage may be recovered by exposing asample of the organ, tissue or cell type to a pilus positive bacterium,such as E. coli K91. In alternative embodiments, phage may be recoveredby amplifying the phage inserts, ligating the inserts to phage DNA andproducing new phage from the ligated DNA.

Proteins and Peptides

In certain embodiments, the present invention concerns novelcompositions comprising at least one protein or peptide. As used herein,a protein or peptide generally refers, but is not limited to, a proteinof greater than about 200 amino acids, up to a full length sequencetranslated from a gene; a polypeptide of greater than about 100 aminoacids; and/or a peptide of from about 3 to about 100 amino acids. Forconvenience, the terms “protein,” “polypeptide” and “peptide are usedinterchangeably herein.

In certain embodiments the size of at least one protein or peptide maycomprise, but is not limited to, 1, 2, 3, 4; 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, about110, about 120, about 130, about 140, about 150, about 160, about 170,about 180, about 190, about 200, about 210, about 220, about 230, about240, about 250, about 275, about 300, about 325, about 350, about 375,about 400, about 425, about 450, about 475, about 500, about 525, about550, about 575, about 600, about 625, about 650, about 675, about 700,about 725, about 750, about 775, about 800, about 825, about 850, about875, about 900, about 925, about 950, about 975, about 1000, about 1100,about 1200, about 1300, about 1400, about 1500, about 1750, about 2000,about 2250, about 2500 or greater amino acid residues.

As used herein, an “amino acid residue” refers to any naturallyoccurring amino acid, any amino acid derivative or any amino acid mimicknown in the art. In certain embodiments, the residues of the protein orpeptide are sequential, without any non-amino acid interrupting thesequence of amino acid residues. In other embodiments, the sequence maycomprise one or more non-amino acid moieties. In particular embodiments,the sequence of residues of the protein or peptide may be interrupted byone or more non-amino acid moieties.

Accordingly, the term “protein or peptide” encompasses amino acidsequences comprising at least one of the 20 common amino acids found innaturally occurring proteins, or at least one modified or unusual aminoacid, including but not limited to those shown on Table 1 below.

TABLE 1 Modified and Unusual Amino Acids Abbr. Amino Acid Aad2-Aminoadipic acid Baad 3-Aminoadipic acid Bala β-alanine,β-Amino-propionic acid Abu 2-Aminobutyric acid 4Abu 4-Aminobutyric acid,piperidinic acid Acp 6-Aminocaproic acid Ahe 2-Aminoheptanoic acid Aib2-Aminoisobutyric acid Baib 3-Aminoisobutyric acid Apm 2-Aminopimelicacid Dbu 2,4-Diaminobutyric acid Des Desmosine Dpm 2,2′-Diaminopimelicacid Dpr 2,3-Diaminopropionic acid EtGly N-Ethylglycine EtAsnN-Ethylasparagine Hyl Hydroxylysine AHyl allo-Hydroxylysine 3Hyp3-Hydroxyproline 4Hyp 4-Hydroxyproline Ide Isodesmosine AIleallo-Isoleucine MeGly N-Methylglycine, sarcosine MeIleN-Methylisoleucine MeLys 6-N-Methyllysine MeVal N-Methylvaline NvaNorvaline Nle Norleucine Orn Ornithine

Proteins or peptides may be made by any technique known to those ofskill in the art, including the expression of proteins, polypeptides orpeptides through standard molecular biological techniques, the isolationof proteins or peptides from natural sources, or the chemical synthesisof proteins or peptides. The nucleotide and protein, polypeptide andpeptide sequences corresponding to various genes have been previouslydisclosed, and may be found at computerized databases known to those ofordinary skill in the art. One such database is the National Center forBiotechnology Information's Genbank and GenPept databases(http://www.ncbi.nlm.nih.gov/). The coding regions for known genes maybe amplified and/or expressed using the techniques disclosed herein oras would be know to those of ordinary skill in the art. Alternatively,various commercial preparations of proteins, polypeptides and peptidesare known to those of skill in the art.

Peptide Mimetics

Another embodiment for the preparation of polypeptides according to theinvention is the use of peptide mimetics. Mimetics arepeptide-containing molecules that mimic elements of protein secondarystructure. See, for example, Johnson et al., “Peptide Turn Mimetics” inBIOTECHNOLOGY AND PHARMACY, Pezzuto et al., Eds., Chapman and Hall, NewYork (1993), incorporated herein by reference. The underlying rationalebehind the use of peptide mimetics is that the peptide backbone ofproteins exists chiefly to orient amino acid side chains in such a wayas to facilitate molecular interactions, such as those of antibody andantigen. A peptide mimetic is expected to permit molecular interactionssimilar to the natural molecule. These principles may be used toengineer second generation molecules having many of the naturalproperties of the targeting peptides disclosed herein, but with alteredand even improved characteristics.

Fusion Proteins

Other embodiments of the present invention concern fusion proteins.These molecules generally have all or a substantial portion of atargeting peptide, linked at the N- or C-terminus, to all or a portionof a second polypeptide or protein. For example, fusions may employleader sequences from other species to permit the recombinant expressionof a protein in a heterologous host. Another useful fusion includes theaddition of an immunologically active domain, such as an antibodyepitope, to facilitate purification of the fusion protein. Inclusion ofa cleavage site at or near the fusion junction will facilitate removalof the extraneous polypeptide after purification. Other useful fusionsinclude linking of functional domains, such as active sites fromenzymes, glycosylation domains, cellular targeting signals ortransmembrane regions. In preferred embodiments, the fusion proteins ofthe instant invention comprise a targeting peptide linked to atherapeutic protein or peptide. Examples of proteins or peptides thatmay be incorporated into a fusion protein include cytostatic proteins,cytocidal proteins, pro-apoptosis agents, anti-angiogenic agents,hormones, cytokines, growth factors, peptide drugs, antibodies, Fabfragments antibodies, antigens, receptor proteins, enzymes, lectins, MHCproteins, cell adhesion proteins and binding proteins. These examplesare not meant to be limiting and it is contemplated that within thescope of the present invention virtually and protein or peptide could beincorporated into a fusion protein comprising a targeting peptide.Methods of generating fusion proteins are well known to those of skillin the art. Such proteins can be produced, for example, by chemicalattachment using bifunctional cross-linking reagents, by de novosynthesis of the complete fusion protein, or by attachment of a DNAsequence encoding the targeting peptide to a DNA sequence encoding thesecond peptide or protein, followed by expression of the intact fusionprotein.

Protein Purification

In certain embodiments a protein or peptide may be isolated or purified.Protein purification techniques are well known to those of skill in theart. These techniques involve, at one level, the homogenization andcrude fractionation of the cells, tissue or organ to polypeptide andnon-polypeptide fractions. The protein or polypeptide of interest may befurther purified using chromatographic and electrophoretic techniques toachieve partial or complete purification (or purification tohomogeneity). Analytical methods particularly suited to the preparationof a pure peptide are ion-exchange chromatography, gel exclusionchromatography, polyacrylamide gel electrophoresis, affinitychromatography, immunoaffinity chromatography and isoelectric focusing.An example of receptor protein purification by affinity chromatographyis disclosed in U.S. Pat. No. 5,206,347, the entire text of which isincorporated herein by reference. A particularly efficient method ofpurifying peptides is fast performance liquid chromatography (FPLC) oreven high performance liquid chromatography (HPLC).

A purified protein or peptide is intended to refer to a composition,isolatable from other components, wherein the protein or peptide ispurified to any degree relative to its naturally-obtainable state. Anisolated or purified protein or peptide, therefore, also refers to aprotein or peptide free from the environment in which it may naturallyoccur. Generally, “purified” will refer to a protein or peptidecomposition that has been subjected to fractionation to remove variousother components, and which composition substantially retains itsexpressed biological activity. Where the term “substantially purified”is used, this designation will refer to a composition in which theprotein or peptide forms the major component of the composition, such asconstituting about 50%, about 60%, about 70%, about 80%, about 90%,about 95%, or more of the proteins in the composition.

Various methods for quantifying the degree of purification of theprotein or peptide are known to those of skill in the art in light ofthe present disclosure. These include, for example, determining thespecific activity of an active fraction, or assessing the amount ofpolypeptides within a fraction by SDS/PAGE analysis. A preferred methodfor assessing the purity of a fraction is to calculate the specificactivity of the fraction, to compare it to the specific activity of theinitial extract, and to thus calculate the degree of purity therein,assessed by a “-fold purification number.” The actual units used torepresent the amount of activity will, of course, be dependent upon theparticular assay technique chosen to follow the purification, andwhether or not the expressed protein or peptide exhibits a detectableactivity.

Various techniques suitable for use in protein purification are wellknown to those of skill in the art. These include, for example,precipitation with ammonium sulphate, PEG, antibodies and the like, orby heat denaturation, followed by: centrifugation; chromatography stepssuch as ion exchange, gel filtration, reverse phase, hydroxylapatite andaffinity chromatography; isoelectric focusing; gel electrophoresis; andcombinations of these and other techniques. As is generally known in theart, it is believed that the order of conducting the variouspurification steps may be changed, or that certain steps may be omitted,and still result in a suitable method for the preparation of asubstantially purified protein or peptide.

There is no general requirement that the protein or peptide always beprovided in their most purified state. Indeed, it is contemplated thatless substantially purified products will have utility in certainembodiments. Partial purification may be accomplished by using fewerpurification steps in combination, or by utilizing different forms ofthe same general purification scheme. For example, it is appreciatedthat a cation-exchange column chromatography performed utilizing an HPLCapparatus will generally result in a greater “−fold” purification thanthe same technique utilizing a low pressure chromatography system.Methods exhibiting a lower degree of relative purification may haveadvantages in total recovery of protein product, or in maintaining theactivity of an expressed protein.

Affinity chromatography is a chromatographic procedure that relies onthe specific affinity between a substance to be isolated and a moleculeto which it can specifically bind. This is a receptor-ligand type ofinteraction. The column material is synthesized by covalently couplingone of the binding partners to an insoluble matrix. The column materialis then able to specifically adsorb the substance from the solution.Elution occurs by changing the conditions to those in which binding willnot occur (e.g., altered pH, ionic strength, temperature, etc.). Thematrix should be a substance that itself does not adsorb molecules toany significant extent and that has a broad range of chemical, physicaland thermal stability. The ligand should be coupled in such a way as tonot affect its binding properties. The ligand should also providerelatively tight binding. And it should be possible to elute thesubstance without destroying the sample or the ligand.

Synthetic Peptides

Because of their relatively small size, the targeting peptides of theinvention can be synthesized in solution or on a solid support inaccordance with conventional techniques. Various automatic synthesizersare commercially available and can be used in accordance with knownprotocols. See, for example, Stewart and Young, Solid Phase PeptideSynthesis, 2d ed. Pierce Chemical Co., 1984; Tam et al., J. Am. Chem.Soc., 105:6442, 1983; Merrifield, Science, 232: 341-347, 1986; andBarany and Merrifield, The Peptides, Gross and Meienhofer, eds.,Academic Press, New York, pp. 1-284, 1979, each incorporated herein byreference. Short peptide sequences, usually from about 6 up to about 35to 50 amino acids, can be readily synthesized by such methods.Alternatively, recombinant DNA technology may be employed wherein anucleotide sequence which encodes a peptide of the invention is insertedinto an expression vector, transformed or transfected into anappropriate host cell, and cultivated under conditions suitable forexpression.

Antibodies

In certain embodiments, it may be desirable to make antibodies againstthe identified targeting peptides or their receptors. The appropriatetargeting peptide or receptor, or portions thereof, may be coupled,bonded, bound, conjugated, or chemically-linked to one or more agentsvia linkers, polylinkers, or derivatized amino acids. This may beperformed such that a bispecific or multivalent composition or vaccineis produced. It is further envisioned that the methods used in thepreparation of these compositions are familiar to those of skill in theart and should be suitable for administration to humans, i.e.,pharmaceutically acceptable. Preferred agents are the carriers arekeyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA).

The term “antibody” is used to refer to any antibody-like molecule thathas an antigen binding region, and includes antibody fragments such asFab′, Fab, F(ab′)₂, single domain antibodies (DABs), Fv, scFv (singlechain Fv), and the like. Techniques for preparing and using variousantibody-based constructs and fragments are well known in the art. Meansfor preparing and characterizing antibodies are also well known in theart (See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, 1988; incorporated herein by reference).

Cytokines and Chemokines

In certain embodiments, it may be desirable to couple specific bioactiveagents to one or more targeting peptides for targeted delivery to anorgan, tissue or cell type. Such agents include, but are not limited to,cytokines, chemokines, pro-apoptosis factors and anti-angiogenicfactors. The term “cytokine” is a generic term for proteins released byone cell population that act on another cell as intercellular mediators.

Examples of such cytokines are lymphokines, monokines, growth factorsand traditional polypeptide hormones. Included among the cytokines aregrowth hormones such as human growth hormone, N-methionyl human growthhormone, and bovine growth hormone; parathyroid hormone; thyroxine;insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such asfollicle stimulating hormone (FSH), thyroid stimulating hormone (TSH),and luteinizing hormone (LH); hepatic growth factor; prostaglandin,fibroblast growth factor; prolactin; placental lactogen, OB protein;tumor necrosis factor-.alpha. and -.beta.; mullerian-inhibitingsubstance; mouse gonadotropin-associated peptide; inhibin; activin;vascular endothelial growth factor; integrin; thrombopoietin (TPO);nerve growth factors such as NGF-.beta.; platelet-growth factor;transforming growth factors (TGFs) such as TGF-.alpha. and TGF-.beta.;insulin-like growth factor-I and -II; erythropoietin (EPO);osteoinductive factors; interferons such as interferon-α, -β, and -γ;colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-1.alpha., IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13, IL-14, IL-15, IL-16,IL-17, IL-18, LIF, G-CSF, GM-CSF, M-CSF, EPO, kit-ligand or FLT-3,angiostatin, thrombospondin, endostatin, tumor necrosis factor and LT.As used herein, the term cytokine includes proteins from natural sourcesor from recombinant cell culture and biologically active equivalents ofthe native sequence cytokines.

Chemokines generally act as chemoattractants to recruit immune effectorcells to the site of chemokine expression. It may be advantageous toexpress a particular chemokine gene in combination with, for example, acytokine gene, to enhance the recruitment of other immune systemcomponents to the site of treatment. Chemokines include, but are notlimited to, RANES, MCAF, MIP1-alpha, MIP1-Beta, and IP-10. The skilledartisan will recognize that certain cytokines are also known to havechemoattractant effects and could also be classified under the termchemokines.

Imaging Agents and Radioisotopes

In certain embodiments, the claimed peptides or proteins of the presentinvention may be attached to imaging agents of use for imaging anddiagnosis of various diseased organs, tissues or cell types. Manyappropriate imaging agents are known in the art, as are methods fortheir attachment to proteins or peptides (see, e.g., U.S. Pat. Nos.5,021,236 and 4,472,509, both incorporated herein by reference). Certainattachment methods involve the use of a metal chelate complex employing,for example, an organic chelating agent such a DTPA attached to theprotein or peptide (U.S. Pat. No. 4,472,509). Proteins or peptides alsomay be reacted with an enzyme in the presence of a coupling agent suchas glutaraldehyde or periodate. Conjugates with fluorescein markers areprepared in the presence of these coupling agents or by reaction with anisothiocyanate.

Non-limiting examples of paramagnetic ions of potential use as imagingagents include chromium (III), manganese (II), iron (III), iron (II),cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III),ytterbium (III), gadolinium (III), vanadium (II), terbium (III),dysprosium (III), holmium (III) and erbium (III), with gadolinium beingparticularly preferred. Ions useful in other contexts, such as X-rayimaging, include but are not limited to lanthanum (III), gold (III),lead (II), and especially bismuth (III).

Radioisotopes of potential use as imaging or therapeutic agents includeastatine²¹¹, ¹⁴carbon, ⁵¹chromium, ³⁶chlorine, ⁵⁷cobalt, ⁵⁸cobalt,copper⁶⁷, ¹⁵²Eu, gallium⁶⁷, ³hydrogen, iodine¹²³, iodine¹²⁵, iodine¹³¹,indium¹¹¹, ⁵⁹iron, ³²phosphorus, rhenium¹⁸⁶, rhenium¹⁸⁸, ⁷⁵selenium,³⁵sulphur, technicium^(99m) and yttrium⁹⁰. ¹²⁵I is often being preferredfor use in certain embodiments, and technicium^(99m) and indium¹¹¹ arealso often preferred due to their low energy and suitability for longrange detection.

Radioactively labeled proteins or peptides of the present invention maybe produced according to well-known methods in the art. For instance,they can be iodinated by contact with sodium or potassium iodide and achemical oxidizing agent such as sodium hypochlorite, or an enzymaticoxidizing agent, such as lactoperoxidase. Proteins or peptides accordingto the invention may be labeled with technetium-^(99m) by ligandexchange process, for example, by reducing pertechnate with stannoussolution, chelating the reduced technetium onto a Sephadex column andapplying the peptide to this column or by direct labeling techniques,e.g., by incubating pertechnate, a reducing agent such as SNCl₂, abuffer solution such as sodium-potassium phthalate solution, and thepeptide. Intermediary functional groups that are often used to bindradioisotopes that exist as metallic ions to peptides arediethylenetriaminepenta-acetic acid (DTPA) and ethylenediaminetetra-acetic acid (EDTA). Also contemplated for use arefluorescent labels, including rhodamine, fluorescein isothiocyanate andrenographin.

In certain embodiments, the claimed proteins or peptides may be linkedto a secondary binding ligand or to an enzyme (an enzyme tag) that willgenerate a colored product upon contact with a chromogenic substrate.Examples of suitable enzymes include urease, alkaline phosphatase,(horseradish) hydrogen peroxidase and glucose oxidase. Preferredsecondary binding ligands are biotin and avidin or streptavidincompounds. The use of such labels is well known to those of skill in theart in light and is described, for example, in U.S. Pat. Nos. 3,817,837;3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241;each incorporated herein by reference.

Cross-Linkers

Bifunctional cross-linking reagents have been extensively used for avariety of purposes including preparation of affinity matrices,modification and stabilization of diverse structures, identification ofligand and receptor binding sites, and structural studies.Homobifunctional reagents that carry two identical functional groupsproved to be highly efficient in inducing cross-linking betweenidentical and different macromolecules or subunits of a macromolecule,and linking of polypeptide ligands to their specific binding sites.Heterobifunctional reagents contain two different functional groups. Bytaking advantage of the differential reactivities of the two differentfunctional groups, cross-linking can be controlled both selectively andsequentially. The bifunctional cross-linking reagents can be dividedaccording to the specificity of their functional groups, e.g., amino,sulfhydryl, guanidino, indole, carboxyl specific groups. Of these,reagents directed to free amino groups have become especially popularbecause of their commercial availability, ease of synthesis and the mildreaction conditions under which they can be applied. A majority ofheterobifunctional cross-linking reagents contains a primaryamine-reactive group and a thiol-reactive group.

Exemplary methods for cross-linking ligands to liposomes are describedin U.S. Pat. No. 5,603,872 and U.S. Pat. No. 5,401,511, eachspecifically incorporated herein by reference in its entirety). Variousligands can be covalently bound to liposomal surfaces through thecross-linking of amine residues. Liposomes, in particular, multilamellarvesicles (MLV) or unilamellar vesicles such as microemulsified liposomes(MEL) and large unilamellar liposomes (LUVET), each containingphosphatidylethanolamine (PE), have been prepared by establishedprocedures. The inclusion of PE in the liposome provides an activefunctional residue, a primary amine, on the liposomal surface forcross-linking purposes. Ligands such as epidermal growth factor (EGF)have been successfully linked with PE-liposomes. Ligands are boundcovalently to discrete sites on the liposome surfaces. The number andsurface density of these sites are dictated by the liposome formulationand the liposome type. The liposomal surfaces may also have sites fornon-covalent association. To form covalent conjugates of ligands andliposomes, cross-linking reagents have been studied for effectivenessand biocompatibility. Cross-linking reagents include glutaraldehyde(GAD), bifunctional oxirane (OXR), ethylene glycol diglycidyl ether(EGDE), and a water soluble carbodiimide, preferably1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). Through thecomplex chemistry of cross-linking, linkage of the amine residues of therecognizing substance and liposomes is established.

In another example, heterobifunctional cross-linking reagents andmethods of using the cross-linking reagents are described (U.S. Pat. No.5,889,155, specifically incorporated herein by reference in itsentirety). The cross-linking reagents combine a nucleophilic hydrazideresidue with an electrophilic maleimide residue, allowing coupling inone example, of aldehydes to free thiols. The cross-linking reagent canbe modified to cross-link various functional groups.

Nucleic Acids

Nucleic acids according to the present invention may encode a targetingpeptide, a receptor protein, a fusion protein or other protein orpeptide. The nucleic acid may be derived from genomic DNA, complementaryDNA (cDNA) or synthetic DNA. Where incorporation into an expressionvector is desired, the nucleic acid may also comprise a natural intronor an intron derived from another gene. Such engineered molecules aresometime referred to as “mini-genes.”

A “nucleic acid” as used herein includes single-stranded anddouble-stranded molecules, as well as DNA, RNA, chemically modifiednucleic acids and nucleic acid analogs. It is contemplated that anucleic acid within the scope of the present invention may be of almostany size, determined in part by the length of the encoded protein orpeptide.

It is contemplated that targeting peptides, fusion proteins andreceptors may be encoded by any nucleic acid sequence that encodes theappropriate amino acid sequence. The design and production of nucleicacids encoding a desired amino acid sequence is well known to those ofskill in the art, using standardized codon tables (see Table 2 below).In preferred embodiments, the codons selected for encoding each aminoacid may be modified to optimize expression of the nucleic acid in thehost cell of interest. Codon preferences for various species of hostcell are well known in the art.

TABLE 2 Amino Acid Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys CUGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAGPhenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine LeuL UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAUProline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGAAGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr TACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAU

In addition to nucleic acids encoding the desired peptide or protein,the present invention encompasses complementary nucleic acids thathybridize under high stringency conditions with such coding nucleic acidsequences. High stringency conditions for nucleic acid hybridization arewell known in the art. For example, conditions may comprise low saltand/or high temperature conditions, such as provided by about 0.02 M toabout 0.15 M NaCl at temperatures of about 50° C. to about 70° C. It isunderstood that the temperature and ionic strength of a desiredstringency are determined in part by the length of the particularnucleic acid(s), the length and nucleotide content of the targetsequence(s), the charge composition of the nucleic acid(s), and to thepresence or concentration of formamide, tetramethylammonium chloride orother solvent(s) in a hybridization mixture.

Vectors for Cloning, Gene Transfer and Expression

In certain embodiments expression vectors are employed to express thetargeting peptide or fusion protein, which can then be purified andused. In other embodiments, the expression vectors are used in genetherapy. Expression requires that appropriate signals be provided in thevectors, and which include various regulatory elements, such asenhancers/promoters from both viral and mammalian sources that driveexpression of the genes of interest in host cells. Elements designed tooptimize messenger RNA stability and translatability in host cells alsoare known.

Regulatory Elements

The terms “expression construct” or “expression vector” are meant toinclude any type of genetic construct containing a nucleic acid codingfor a gene product in which part or all of the nucleic acid codingsequence is capable of being transcribed. In preferred embodiments, thenucleic acid encoding a gene product is under transcriptional control ofa promoter. A “promoter” refers to a DNA sequence recognized by thesynthetic machinery of the cell, or introduced synthetic machinery,required to initiate the specific transcription of a gene. The phrase“under transcriptional control” means that the promoter is in thecorrect location and orientation in relation to the nucleic acid tocontrol RNA polymerase initiation and expression of the gene.

The particular promoter employed to control the expression of a nucleicacid sequence of interest is not believed to be important, so long as itis capable of directing the expression of the nucleic acid in thetargeted cell. Thus, where a human cell is targeted, it is preferable toposition the nucleic acid coding region adjacent and under the controlof a promoter that transcriptionally active in human cells. Generallyspeaking, such a promoter might include either a human or viralpromoter.

In various embodiments, the human cytomegalovirus (CMV) immediate earlygene promoter, the SV40 early promoter, the Rouse sarcoma virus longterminal repeat, rat insulin promoter, and glyceraldehyde-3-phosphatedehydrogenase promoter can be used to obtain high-level expression ofthe coding sequence of interest. The use of other viral or mammaliancellular or bacterial phage promoters that are well-known in the art toachieve expression of a coding sequence of interest is contemplated aswell, provided that the levels of expression are sufficient for a givenpurpose.

Where a cDNA insert is employed, one will typically include apolyadenylation signal to effect proper polyadenylation of the genetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the invention, and any suchsequence may be employed, such as human growth hormone and SV40polyadenylation signals. Also contemplated as an element of theexpression construct is a terminator. These elements can serve toenhance message levels and to minimize read through from the constructinto other sequences.

Selectable Markers

In certain embodiments of the invention, the cells containing nucleicacid constructs of the present invention may be identified in vitro orin vivo by including a marker in the expression construct. Such markerswould confer an identifiable change to the cell permitting easyidentification of cells containing the expression construct. Usually theinclusion of a drug selection marker aids in cloning and in theselection of transformants. For example, genes that confer resistance toneomycin, puromycin, hygromycin, DHFR, GPT, zeocin, and histidinol areuseful selectable markers. Alternatively, enzymes such as herpes simplexvirus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT)may be employed. Immunologic markers also can be employed. Theselectable marker employed is not believed to be important, so long asit is capable of being expressed simultaneously with the nucleic acidencoding a gene product. Further examples of selectable markers are wellknown to one of skill in the art.

Delivery of Expression Vectors

There are a number of ways in which expression vectors may introducedinto cells. In certain embodiments of the invention, the expressionconstruct comprises a virus or engineered construct derived from a viralgenome. The ability of certain viruses to enter cells viareceptor-mediated endocytosis, to integrate into host cell genome, andexpress viral genes stably and efficiently have made them attractivecandidates for the transfer of foreign genes into mammalian cells(Ridgeway, 1988; Nicolas and Rubinstein, In: Vectors: A survey ofmolecular cloning vectors and their uses, Rodriguez and Denhardt, eds.,Stoneham: Butterworth, pp. 494-513, 1988.; Baichwal and Sugden,Baichwal, In: Gene Transfer, Kucherlapati R, ed., New. York, PlenumPress, pp. 117-148, 1986. 1986; Temin, In: Gene Transfer, Kucherlapati,R. ed., New York, Plenum Press, pp. 149-188, 1986). Preferred genetherapy vectors are generally viral vectors.

In using viral delivery systems, one will desire to purify the virionsufficiently to render it essentially free of undesirable contaminants,such as defective interfering viral particles or endotoxins and otherpyrogens such that it will not cause any untoward reactions in the cell,animal or individual receiving the vector construct. A preferred meansof purifying the vector involves the use of buoyant density gradients,such as cesium chloride gradient centrifugation.

DNA viruses used as gene vectors include the papovaviruses (e.g., simianvirus 40, bovine papilloma virus, and polyoma) (Ridgeway, pp 467-492,1988; Baichwal and Sugden, 1986) and adenoviruses (Ridgeway, 1988;Baichwal and Sugden, 1986).

One of the preferred methods for in vivo delivery involves the use of anadenovirus expression vector. Although adenovirus vectors are known tohave a low capacity for integration into genomic DNA, this feature iscounterbalanced by the high efficiency of gene transfer afforded bythese vectors. “Adenovirus expression vector” is meant to include, butis not limited to, constructs containing adenovirus sequences sufficientto (a) support packaging of the construct and (b) to express anantisense or a sense polynucleotide that has been cloned therein.

Generation and propagation of adenovirus vectors that are replicationdeficient depend on a unique helper cell line, designated 293, which istransformed from human embryonic kidney cells by Ad5 DNA fragments andconstitutively expresses E1 proteins (Graham et al., J. Gen. Virol.,36:59-72, 1977.). Since the E3 region is dispensable from the adenovirusgenome (Jones and Shenk, Cell, 13:181-188, 1978), the current adenovirusvectors, with the help of 293 cells, carry foreign DNA in either the E1,the E3, or both regions (Graham and Prevec, In: Methods in MolecularBiology: Gene Transfer and Expression Protocol, E. J. Murray, ed.,Humana Press, Clifton, N.J., 7:109-128, 1991.).

Helper cell lines may be derived from human cells such as humanembryonic kidney cells, muscle cells, hematopoietic cells or other humanembryonic mesenchymal or epithelial cells. Alternatively, the helpercells may be derived from the cells of other mammalian species that arepermissive for human adeno virus. Such cells include, e.g., Vero cellsor other monkey embryonic mesenchymal or epithelial cells. As discussed,the preferred helper cell line is 293. Racher et al., (Biotechnol. Tech.9:169-174, 1995) disclosed improved methods for culturing 293 cells andpropagating adenovirus.

Adenovirus vectors have been used in eukaryotic gene expression (Levreroet al., Gene, 101:195-202, 1991; Gomez-Foix et al., J. Biol. Chem.,267:25129-25134, 1992) and vaccine development (Grunhaus and Horwitz,1992; Graham and Prevec, 1991). Animal studies have suggested thatrecombinant adenovirus could be used for gene therapy(Stratford-Perricaudet and Perricaudet, In: Human Gene Transfer, O.Cohen-Haguenauer et al, eds. John Libbey Eurotext, France, pp. 51-61,1991; Stratford-Perricaudet et al., Hum. Gene Ther. 1:241-256, 1990;Rich et al., Hum. Gene. Ther. 4:461-476, 1993). Studies in administeringrecombinant adenovirus to different tissues include trachea instillation(Rosenfeld et al., Science, 252: 431-434, 1991; Rosenfeld et al., Cell,68: 143-155, 1992), muscle injection (Ragot et al., Nature, 361:647-650,1993), peripheral intravenous injections (Herz and Gerard, Proc. Natl.Acad. Sci. USA, 90:2812-2816, 1993) and stereotactic innoculation intothe brain (Le Gal La Salle et al., Science, 259:988-990, 1993).

Other gene transfer vectors may be constructed from retroviruses.(Coffin, In: Virology, Fields et al., eds., Raven Press, New York, pp.1437-1500, 1990.) The retroviral genome contains three genes, gag, pol,and env. that code for capsid proteins, polymerase enzyme, and envelopecomponents, respectively. A sequence found upstream from the gag genecontains a signal for packaging of the genome into virions. Two longterminal repeat (LTR) sequences are present at the 5′ and 3′ ends of theviral genome. These contain strong promoter and enhancer sequences, andalso are required for integration in the host cell genome (Coffin,1990).

In order to construct a retroviral vector, a nucleic acid encodingprotein of interest is inserted into the viral genome in the place ofcertain viral sequences to produce a virus that isreplication-defective. In order to produce virions, a packaging cellline containing the gag, pol, and env genes, but without the LTR andpackaging components, is constructed (Mann et al., Cell, 33:153-159,1983). When a recombinant plasmid containing a cDNA, together with theretroviral LTR and packaging sequences is introduced into this cell line(by calcium phosphate precipitation for example), the packaging sequenceallows the RNA transcript of the recombinant plasmid to be packaged intoviral particles, which are then secreted into the culture media (Nicolasand Rubenstein, 1988; Temin, 1986; Mann et al., 1983). The mediacontaining the recombinant retroviruses is then collected, optionallyconcentrated, and used for gene transfer. Retroviral vectors are capableof infecting a broad variety of cell types. However, integration andstable expression require the division of host cells (Paskind et al.,Virology, 67:242-248, 1975).

Other viral vectors may be employed as expression constructs. Vectorsderived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwaland Sugden, 1986; Coupar et al., Gene 68:1-10, 1988), adeno-associatedvirus (AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986; Hermonat andMuzycska, Proc. Natl. Acad. Sci. USA, 81: 6466-6470, 1984), and herpesviruses may be employed. They offer several attractive features forvarious mammalian cells (Friedmann, Science, 244:1275-1281, 1989;Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988; Horwichet al., J. Virol., 64:642-650, 1990).

Several non-viral methods for the transfer of expression constructs intocultured mammalian cells also are contemplated by the present invention.These include calcium phosphate precipitation (Graham and van der Eb,Virology, 52:456-467, 1973.; Chen and Okayama, Mol. Cell. Biol.,7:2745-2752, 1987.; Rippe et al., Mol. Cell. Biol. 10: 689-695, 1990;DEAE dextran (Gopal, et al. Mol. Cell. Biol., 5:1188-1190, 1985),electroporation (Tur-Kaspa et al., Mol. Cell. Biol., 6:716-718, 1986;Potter et al., Proc. Natl. Acad. Sci. USA, 81: 7161-7165, 1984), directmicroinjection, DNA-loaded liposomes and lipofectamine-DNA complexes,cell sonication, gene bombardment using high velocity microprojectiles,and receptor-mediated transfection (Wu and Wu, J. Biol. Chem.262:4429-4432, 1987; Wu and Wu, Biochemistry, 27:887-892, 1988). Some,of these techniques may be successfully adapted for in vivo or ex vivouse.

In a further embodiment of the invention, the expression construct maybe entrapped in a liposome. Liposome-mediated nucleic acid delivery andexpression of foreign DNA in vitro has been very successful. Wong etal., (Gene, 10:87-94, 1980) demonstrated the feasibility ofliposome-mediated delivery and expression of foreign DNA in culturedchick embryo, HeLa, and hepatoma cells. Nicolau et al., (MethodsEnzymol., 149:157-176, 1987.) accomplished successful liposome-mediatedgene transfer in rats after intravenous injection.

Pharmaceutical Compositions

Where clinical applications are contemplated, it may be necessary toprepare pharmaceutical compositions—expression vectors, virus stocks,proteins, antibodies and drugs—in a form appropriate for the intendedapplication. Generally, this will entail preparing compositions that areessentially free of impurities that could be harmful to humans oranimals.

One generally will desire to employ appropriate salts and buffers torender delivery vectors stable and allow for uptake by target cells.Buffers also are employed when recombinant cells are introduced into apatient. Aqueous compositions of the present invention may comprise aneffective amount of a protein, peptide, fusion protein, recombinantphage and/or expression vector, dissolved or dispersed in apharmaceutically acceptable carrier or aqueous medium. Such compositionsalso are referred to as innocula. The phrase “pharmaceutically orpharmacologically acceptable” refers to molecular entities andcompositions that do not produce adverse, allergic, or other untowardreactions when administered to an animal or a human. As used herein,“pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents and the like. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the proteins or peptides of the present invention, itsuse in therapeutic compositions is contemplated. Supplementary activeingredients also can be incorporated into the compositions.

The active compositions of the present invention may include classicpharmaceutical preparations. Administration of these compositionsaccording to the present invention are via any common route so long asthe target tissue is available via that route. This includes oral,nasal, buccal, rectal, vaginal or topical. Alternatively, administrationmay be by orthotopic, intradermal, subcutaneous, intramuscular,intraperitoneal, intraarterial or intravenous injection. Suchcompositions normally would be administered as pharmaceuticallyacceptable compositions, described supra.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it is preferable to include isotonic agents,for example, sugars or sodium chloride. Prolonged absorption of theinjectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousother ingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating thevarious sterilized active ingredients into a sterile vehicle whichcontains the basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum-drying and freeze-drying techniques which yield apowder of the active ingredient plus any additional desired ingredientfrom a previously sterile-filtered solution thereof.

Therapeutic Agents

In certain embodiments, therapeutic agents may be attached to atargeting peptide or fusion protein for selective delivery to, forexample, white adipose tissue. Agents or factors suitable for use mayinclude any chemical compound that induces apoptosis, cell death, cellstasis and/or anti-angiogenesis.

Regulators of Programmed Cell Death

Apoptosis, or programmed cell death, is an essential process for normalembryonic development, maintaining homeostasis in adult tissues, andsuppressing carcinogenesis (Kerr et al., 1972). The Bcl-2 family ofproteins and ICE-like proteases have been demonstrated to be importantregulators and effectors of apoptosis in other systems. The Bcl-2protein, discovered in association with follicular lymphoma, plays aprominent role in controlling apoptosis and enhancing cell survival inresponse to diverse apoptotic stimuli (Bakhshi et al., 1985; Cleary andSklar, 1985; Cleary et al., 1986; Tsujimoto et al., 1985; Tsujimoto andCroce, 1986). The evolutionarily conserved Bcl-2 protein now isrecognized to be a member of a family of related proteins, which can becategorized as death agonists or death antagonists.

Subsequent to its discovery, it was shown that Bcl-2 acts to suppresscell death triggered by a variety of stimuli. Also, it now is apparentthat there is a family of Bcl-2 cell death regulatory proteins thatshare in common structural and sequence homologies. These differentfamily members have been shown to either possess similar functions toBcl-2 (e.g., Bcl_(XL), Bcl_(W), Bcl_(S), Mcl-1, A1, Bfl-1) or counteractBcl-2 function and promote cell death (e.g., Bax, Bak, Bik, Bim, Bid,Bad, Harakiri).

Non-limiting examples of pro-apoptosis agents contemplated within thescope of the present invention include gramicidin, magainin, mellitin,defensin, cecropin, (KLAKLAK)₂ (SEQ ID NO:1), (KLAKKLA)₂ (SEQ ID NO:2),(KAAKKAA)₂ (SEQ ID NO:3) or (KLGKKLG)₃ (SEQ ID NO:4).

Angiogenic Inhibitors

In certain embodiments the present invention may concern administrationof targeting peptides attached to anti-angiogenic agents, such asangiotensin, laminin peptides, fibronectin peptides, plasminogenactivator inhibitors, tissue metalloproteinase inhibitors, interferons,interleukin 12, platelet factor 4, IP-10, Gro-β, thrombospondin,2-methoxyoestradiol, proliferin-related protein, carboxiamidotriazole,CM101, Marimastat, pentosan polysulphate, angiopoietin 2 (Regeneron),interferon-alpha, herbimycin A, PNU145156E, 16K prolactin fragment,Linomide, thalidomide, pentoxifylline, genistein, TNP470, endostatin,paclitaxel, accutin, angiostatin, cidofovir, vincristine, bleomycin,AGM-1470, platelet factor 4 or minocycline.

Proliferation of tumors cells relies heavily on extensive tumorvascularization, which accompanies cancer progression. Thus, inhibitionof new blood vessel formation with anti-angiogenic agents and targeteddestruction of existing blood vessels have been introduced as aneffective and relatively non-toxic approach to tumor treatment. (Arap etal., Science 279:377-380, 1998; Arap et al., Curr. Opin. Oncol.10:560-565, 1998; Ellerby et al., Nature Med. 5:1032-1038, 1999). Avariety of anti-angiogenic agents and/or blood vessel inhibitors areknown. (E.g., Folkman, In: Cancer: Principles and Practice, eds. DeVitaet al., pp. 3075-3085, Lippincott-Raven, New York, 1997; Eliceiri andCheresh, Curr. Opin. Cell. Biol. 13, 563-568, 2001).

White fat represents a unique tissue that, like tumors, can quicklyproliferate and expand (Wasserman, In: Handbook of Physiology, eds.Renold and Cahill, pp. 87-100, American Physiological Society,Washington, D.C., 1965; Cinti, Eat. Weight. Disord. 5:132-142, 2000).Studies of adipose tissue reveal that it is highly vascularized.Multiple capillaries make contacts with every adipocyte, suggesting theimportance of the vasculature for maintenance of the fat mass (Crandallet al., Microcirculation 4:211-232, 1997). A hypothesis underlying thepresent invention is that adipose tissue proliferation might rely onangiogenesis similarly to tumors. If so, destruction of fatneovasculature could prevent the development of obesity, whereastargeting existing adipose blood vessels could potentially result in fatregression. Methods of use of adipose targeting peptides may includeinduction of weight loss, treatment of obesity and/or treatment of HIVrelated lipodystrophy.

Cytotoxic Agents

Chemotherapeutic (cytotoxic) agents of potential use include, but arenot limited to, 5-fluorouracil, bleomycin, busulfan, camptothecin,carboplatin, chlorambucil, cisplatin (CDDP), cyclophosphamide,dactinomycin, daunorubicin, doxorubicin, estrogen receptor bindingagents, etoposide (VP16), farnesyl-protein transferase inhibitors,gemcitabine, ifosfamide, mechlorethamine, melphalan, mitomycin,navelbine, nitrosurea, plicomycin, procarbazine, raloxifene, tamoxifen,taxol, temazolomide (an aqueous form of DTIC), transplatinum,vinblastine and methotrexate, vincristine, or any analog or derivativevariant of the foregoing. Most chemotherapeutic agents fall into thecategories of alkylating agents, antimetabolites, antitumor antibiotics,corticosteroid hormones, mitotic inhibitors, and nitrosoureas, hormoneagents, miscellaneous agents, and any analog or derivative variantthereof.

Chemotherapeutic agents and methods of administration, dosages, etc. arewell known to those of skill in the art (see for example, the“Physicians Desk Reference”, Goodman & Gilman's “The PharmacologicalBasis of Therapeutics” and in “Remington's Pharmaceutical Sciences”15^(th) ed., pp 1035-1038 and 1570-1580, incorporated herein byreference in relevant parts), and may be combined with the invention inlight of the disclosures herein. Some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject. Examples ofspecific chemotherapeutic agents and dose regimes are also describedherein. Of course, all of these dosages and agents described herein areexemplary rather than limiting, and other doses or agents may be used bya skilled artisan for a specific patient or application. Any dosagein-between these points, or range derivable therein is also expected tobe of use in the invention.

Alkylating Agents

Alkylating agents are drugs that directly interact with genomic DNA toprevent cells from proliferating. This category of chemotherapeuticdrugs represents agents that affect all phases of the cell cycle, thatis, they are not phase-specific. An alkylating agent, may include, butis not limited to, a nitrogen mustard, an ethylenimene, amethylmelamine, an alkyl sulfonate, a nitrosourea or a triazines. Theyinclude but are not limited to: busulfan, chlorambucil, cisplatin,cyclophosphamide (cytoxan), dacarbazine, ifosfamide, mechlorethamine(mustargen), and melphalan.

Antimetabolites

Antimetabolites disrupt DNA and RNA synthesis. Unlike alkylating agents,they specifically influence the cell cycle during S phase.Antimetabolites can be differentiated into various categories, such asfolic acid analogs, pyrimidine analogs and purine analogs and relatedinhibitory compounds. Antimetabolites include but are not limited to,5-fluorouracil (5-FU), cytarabine (Ara-C), fludarabine, gemcitabine, andmethotrexate.

Natural Products

Natural products generally refer to compounds originally isolated from anatural source, and identified as having a pharmacological activity.Such compounds, analogs and derivatives thereof may be, isolated from anatural source, chemically synthesized or recombinantly produced by anytechnique known to those of skill in the art. Natural products includesuch categories as mitotic inhibitors, antitumor antibiotics, enzymesand biological response modifiers.

Mitotic inhibitors include plant alkaloids and other natural agents thatcan inhibit either protein synthesis required for cell division ormitosis. They operate during a specific phase during the cell cycle.Mitotic inhibitors include, for example, docetaxel, etoposide (VP16),teniposide, paclitaxel, taxol, vinblastine, vincristine, andvinorelbine.

Taxoids are a class of related compounds isolated from the bark of theash tree, Taxus brevifolia. Taxoids include but are not limited tocompounds such as docetaxel and paclitaxel. Paclitaxel binds to tubulin(at a site distinct from that used by the vinca alkaloids) and promotesthe assembly of microtubules.

Vinca alkaloids are a type of plant alkaloid identified to havepharmaceutical activity. They include such compounds as vinblastine(VLB) and vincristine.

Antibiotics

Certain antibiotics have both antimicrobial and cytotoxic activity.These drugs also interfere with DNA by chemically inhibiting enzymes andmitosis or altering cellular membranes. These agents are not phasespecific so they work in all phases of the cell cycle. Examples ofcytotoxic antibiotics include, but are not limited to, bleomycin,dactinomycin, daunorubicin, doxorubicin (Adriamycin), plicamycin(mithramycin) and idarubicin.

Miscellaneous Agents

Miscellaneous cytotoxic agents that do not fall into the previouscategories include, but are not limited to, platinum coordinationcomplexes, anthracenediones, substituted ureas, methyl hydrazinederivatives, amsacrine, L-asparaginase, and tretinoin. Platinumcoordination complexes include such compounds as carboplatin andcisplatin (cis-DDP). An exemplary anthracenedione is mitoxantrone. Anexemplary substituted urea is hydroxyurea. An exemplary methyl hydrazinederivative is procarbazine (N-methylhydrazine, MIH). These examples arenot limiting and it is contemplated that any known cytotoxic, cytostaticor cytocidal agent may be attached to targeting peptides andadministered to a targeted organ, tissue or cell type within the scopeof the invention.

Dosages

The skilled artisan is directed to “Remington's Pharmaceutical Sciences”15th Edition, chapter 33, and in particular to pages 624-652. Somevariation in dosage will necessarily occur depending on the condition ofthe subject being treated. The person responsible for administrationwill, in any event, determine the appropriate dose for the individualsubject. Moreover, for human administration, preparations should meetsterility, pyrogenicity, and general safety and purity standards asrequired by the FDA Office of Biologics standards.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Identification of Mouse Placenta Targeting Peptides

Identification of Placenta Homing Peptides

Peptides homing to the mouse placenta were identified by a post-clearingprotocol using a phage display library. A first round of biopanning wasperformed on pregnant mice. Samples of placenta were removed and phagerescued according to protocols described below, with one modification.In the typical biopanning protocol, thousands of phage may be recoveredfrom a single organ, tissue or cell type. Typically, between 200 and 300individual colonies are selected from plated phage and these areamplified and pooled to form the phage display library for the second orthird rounds of biopanning. In the present Example, all phage rescuedfrom the first round of biopanning were amplified in bulk on solidmedium and then pooled to form the phage display library for the secondround of biopanning. That is, there was no restriction of the rescuedphage from the first round of biopanning. This in vivo biopanningwithout restriction was performed for three-rounds (rounds I-III), thena post-clearing procedure was used.

In a post-clearing protocol (round IV), phage were administered to anon-pregnant mouse. Phage that bound to tissues other than placenta wereabsorbed from the circulation. Remaining phage were recovered from theplasma of the non-pregnant mouse. This protocol was designed to isolatephage that bound to placenta but not to other mouse organs, tissues orcell types. The following placenta-targeting peptides were identified,along with their frequencies. A search of the GenBank database disclosedthat none of the sequences listed below was 100% homologous with anyknown peptide sequence.

(SEQ ID NO:5) TPKTSVT 7.4% in round III, 8.5% in round IV (SEQ ID NO:6)RMDGPVR 3.1% in round III, 8.5% in round IV (SEQ ID NO:7) RAPGGVR <1% inround III, 8.5% in round IV (SEQ ID NO:8) VGLHARA 4.2% in round III,7.4% in round IV (SEQ ID NO:9) YIRPFTL 2.1% in round III, 5.3% in roundIV (SEQ ID NO:10) LGLRSVG <1% in round III, 5.3% in round IV (SEQ IDNO:11) PSERSPS (data not available)

As can be seen, the use of a post-clearing procedure resulted in asubstantial enrichment of phage bearing placenta targeting peptides.Although this procedure was used for placenta, the skilled artisan willrealize that post-clearance can be performed for any organ, tissue orcell type where a phage library can be administered to a subject lackingthat organ, tissue or cell type. For example, a post-clearing procedurefor prostate or testicle targeting peptides could be performed in afemale subject, and for ovary, vagina or uterus in a male subject.

A homology search identified several candidate proteins as endogenousanalogs of the placental targeting peptides, including TCR gamma-1(TPKTSVT, SEQ ID NO:5), tenascin (RMDGPVR, SEQ ID NO:6 and RAPGGVR, SEQID NO:7), angiotensin I (YIRPFTL, SEQ ID NO:9) and MHC H2-D-q alphachain (VGLHARA, SEQ ID NO:8).

Validation of Placenta Homing Peptides and Inhibition of Pregnancy

The placenta homing peptides were validated in vivo by injection intopregnant mice and recovery from the placenta. FIG. 1 shows the resultsof the validation studies for selected placenta homing phage. The phageclones are identified as: PA—TPKTSVT (SEQ ID NO:5), PC—RAPGGVR (SEQ IDNO:7), PE—LGLRSVG (SEQ ID NO:10), PF—YIRPFTL (SEQ ID NO:9). It can beseen that the PA clone exhibited placental homing more than an order ofmagnitude greater than observed with control fd-tet phage. The PC clonealso showed substantially higher placental localization, while the PEand PF clones were not substantially enriched in placenta compared tocontrol phage.

Despite the absence of apparent enrichment of the PF clone in placentaltissue, both the PA and PF peptides showed anti-placental activity.Table 3 shows the effects of the PA and PF placental targeting peptidesinjected into pregnant mice, attached to FITC (fluoresceinisothiocyanate), GST (glutathion S-transferase) or to phage. At lowerdosages (450 μg total), FITC conjugated PA and PF showed a slight effecton pregnancy (Table 3). At higher dosages (800 to 1000 μg protein or4.5×10¹⁰ phage), both protein and phage conjugated PA and PF peptidessubstantially interfered with fetal development (Table 3), apparentlyresulting in death of the fetuses in most cases. The CARAC peptide (SEQID NO:12), an adipose targeting peptide (FE, TREVHRS, SEQ ID NO:13) orfd-tet phage were used as non-placental targeting controls.

TABLE 3 Effect of Placental Targeting Peptides on Fetal DevelopmentPeptide Injected Pregnancy Outcome Peptide Effect on Embryo Inhibitionwith FITC conjugates -I 1 mouse injected iv (predominantly) or ip ~everyother day, day 1-day 18, 9 times, Total 450 mM (~450 μg) CARAC-FITC (-control) Delivery: 18 d, 5 normal pups No effect PA-FITC (placentahomer) Delivery: 19 d, 8 normal pups No effect PF-FITC (placenta homer)Delivery: 21 d, 1 dead pup Development delay, toxicity Inhibition withFITC conjugates -II 1 mouse injected sc (predominantly) or iv ~everyother day, day 4-day 17, 10 times, Total 1 M (~1 mg) CARAC-FITC (-control) Delivery: 20 d, 5 pups, 1-dead Slight toxicity? PA-FITC(placenta homer) No fetuses inside after 21 d Pregnancy terminationPF-FITC (placenta homer) No fetuses inside after 21 d Pregnancytermination Inhibition with phage conjugates -I 1 mouse injected iv(predominantly) or ip ~every other day, day 1-day 18, 9 times, Total 45× 10¹⁰ TU Fd-Tet (- control) Avertin OD => death. fetuses-OK ? PA-phage(placenta homer) Delivery: 24 d, 4 pups, 1-dead Development delay,toxicity PF-phage (placenta homer) Delivery: 25 d, 8 pups, all deadDevelopment delay, toxicity Inhibition with GST conjugates -I 1 mouseinjected sc (predominantly) or iv ~every other day, day 4-day 17, 10times, Total 800 μg GST-FE (- control) Delivery: 20 d, 2 pups, OK Noeffect GST-PA (placenta homer) No delivery or fetuses after 21 dPregnancy termination GST-PF (placenta homer) Day 15: no fetuses inside,uterus necrotic Pregnancy termination

These results validate the placental targeting peptide sequencesidentified above. They further demonstrate that even in the absence ofsubstantial enrichment of phage bearing the targeting sequence to thetarget organ (e.g. peptide PF, FIG. 1), the targeting peptide maynevertheless provide for targeted delivery of therapeutic agents to thetarget organ. In this study, it appeared that at lower dosages the PFpeptide was more effective than the PA peptide at interfering withpregnancy, despite the observation that the PA peptide produced amany-fold higher level of phage localization to placenta.

The skilled artisan will realize that the disclosed methods and peptidesmay be of use for targeted delivery of therapeutic agents to the fetusthrough the placenta, as well as for novel approaches to terminatingpregnancy and/or inducing labor.

Example 2 Localization of the TPKTSVT (SEQ ID NO:5) Peptide in MousePlacenta

Material and Methods

Animals Staged pregnant 18 days postconception (dpc) C57BIJ6 female micewere purchased from Harlan Teklad (Indianapolis, Ind.). Congenicpregnant β2m-null females (stock 002087) mice were purchased from TheJackson Laboratories (Bar Harbor, Me.). Anesthesia was performed withAvertin (0.015 ml/g) administered intraperitoneally (Pasqualini andRuoslahti, 1996, Nature 380:364-366; Rajotte et al., 1998, J. Clin.Invest. 102:430-437).

Phage Library Screening In vivo screening of an M13 page-display CX₇Clibrary (Pasqualini et al., 2000, in Phage Display: A Laboratory Manual,eds. Barbas et al., Cold Spring Harbor Laboratory Press, New York, N.Y.,pp. 22.1-24; Arap et al., 2002, Nature Med. 8:121-127), forplacenta-homing peptides was performed as described (Pasqualini et al.,2000; Pasqualini and Ruoslahti, 1996) with novel modifications describedbelow. In each biopanning round, an 18 dpc C57BL/6 female was injectedintravenously (tail vein) with 10¹⁰ transducing units (TU) of thelibrary. Increasing amounts of phage (from ˜10³ TU in round 1 to ˜10⁴ TUin round 4) were recovered from the placentas after 5 min ofcirculation. Recovered phage were bulk-amplified for subsequent roundsof screening. In a procedure introduced here for the first time, thesub-library that was amplified after the third round of panning wascleared of nonspecific binders in a subtraction step. A virgin C57BL/6female was infused through the tail vein with 10⁹ TU of phage selectedin round 3. After 5 min, the unbound circulating phage were recoveredfrom plasma. The plasma contained approximately 10⁷ TU of pre-clearedphage. The precleared phage population, representing less than 1% of theinjected pool, was recovered and amplified for the final round ofbiopanning.

Phage Recovery Mouse placentas and embryonic livers were individuallyweighed, ground with a glass Dounce homogenizer and suspended in 1 ml ofDulbecco's Modified Eagle's Medium (DMEM) containing proteinaseinhibitors (DMEM-prin—1 mM PMSF, 20 μg/ml aprotinin, and 1 μg/mlleupeptin). The suspension was vortexed and washed three times withDMEM-prin. Tissue homogenates (or 10 ml of blood for normalization ofphage titer in placenta against circulating phage titer) were incubatedwith 1 ml of host bacteria (log phase E. coli K91kan; OD600 ˜2).Aliquots of the bacterial culture were plated onto Luria-Bertani agarplates containing 40 μg/ml tetracycline and 100 μg/ml kanamycin. Plateswere incubated overnight at 37° C. Triplicate samples were processed forhost bacterial infection, phage recovery, and histological analysis.

Fusion and Recombinant Peptides Carboxyfluorescein (FrrC)-conjugatedCTPKTSVTC (SEQ ID NO:144) or control peptide CARAC (SEQ ID NO:12),formed into cyclic peptides using the flanking cysteines, werechemically synthesized and HPLC-purified to >90% purity by Anaspec (SanJose, Calif.). The FITC-peptide stocks were made by dissolvinglyophilized peptides in DMSO to a concentration of 20 mM, after whichthe peptides were diluted to 1 mM with PBS (phosphate buffered saline)and aliquots were frozen until use. The CTPKTSVTC (SEQ ID NO:144)peptide and an unrelated control peptide, CTREVHRSC (SEQ ID NO:87),fused in-frame with GST at the amino-terminus were purified toapproximately 90% purity using the BugBuster GST Bind Kit (Novagen,Madison, Wis.). Purified peptides were buffer-exchanged into PBS usingCentricon PL-10 columns (Millipore, Bedford, Mass.) and the aliquotswere frozen until use. For in vivo peptide homing validation, 10 μl ofFITC-peptide stocks diluted 20-fold with PBS or, 250 μl of 5 mg mlGST-peptides were injected. For phage homing competition and IgGtranscytosis blocking experiments, 500 μl of 5 mg/ml GST-peptides wereadministered intravenously.

Peptide Localization in Tissues Immunohistochemistry on sections offormalin-fixed, paraffin-embedded mouse tissue was performed asdescribed (Pasqualini et al., 2000; Pasqualini and Ruoslahti, 1996). Forphage-peptide immunolocalization, a rabbit anti-fd phage antibody (SigmaChemicals, St. Louis, Mo.) was used at 1:1,000 dilution and detectedwith a secondary horseradish peroxidase (HRP)-conjugated antibody. ForGST-peptide immunolocalization, a goat anti-GST antibody (Amersham,Piscataway, N.J.) was used at 1:1,000 dilution and detected with asecondary alkaline phosphatase (AP)-conjugated antibody. For mouse IgGiminunolocalization, the ARK Peroxidase Kit (DAKO, Carpinteria, CA) wasused. All immunohistochemistry and FITC immunofluorescence images werecaptured using an Olympus IX70 microscope and digital camera setup.

Peptide Embrotoxicity For peptide embryotoxicity studies, agents wereinjected at the following daily doses: GST-peptides 0.1 mg (˜3 nMoles),FITC-peptides 50 μg (˜30 nMoles), and phage-peptides 10¹¹ TU. All agentswere dissolved in PBS. Mice were injected subcutaneously in the back(5-10 injections per course).

Results

Localization of the TPKTSVT (SEQ ID NO:5) Peptide in Mouse Placenta Thetissue distribution of recombinant phage injected into pregnant mice wasexamined by immunohistochemistry (FIG. 3). While a control phage barelylocalized to placental tissues (FIG. 3A), the TPKTSVT (SEQ ID NO:5)phage homed to the placenta and showed marked localization to the villiof the visceral yolk sac (vys) endoderm (FIG. 3B, arrows). The vys is alayer of epithelial cells which surrounds the embryonic microvasculatureand functions as the final barrier during transport of molecules, suchas immunoglobulin G (IgG), from the labyrinth layer of the placenta intothe fetus (Rugh, 1990; Beckman et al., 1990; Lyden et al., 2001, J.Immunol. 166:3882-3889; Jollie, 1990, Teratology 41:361-381).

To verify that targeting of the TPKTSVT (SEQ ID NO:5) motif to the vysendoderm also occurs if the peptide is outside of the context of thephage, the homing of TPKTSVT (SEQ ID NO:5) fused with glutathioneS-transferase (GST) protein to placenta was also tested. GST fused toeither TPKTSVT (SEQ ID NO:5) or a control peptide TREVHRS (SEQ ID NO:13)with a similar overall charge were injected into pregnant mice and thetissue distribution of each peptide was examined. No localization of thecontrol GST fusion peptide to the placenta was observed (FIG. 3C). Incontrast, accumulation of the TPKTSVT (SEQ ID NO:5) GST fusion peptidewas readily detectable in the apical cytoplasm of the vys (FIG. 3D,arrows) and matched that observed for TPKTSVT (SEQ ID NO:5)-phage (FIG.3B, arrows).

Similarly, fluorescein (FITC)-conjugated TPKTSVT (SEQ ID NO:5) injectedintravenously into pregnant mice also specifically localized to theapical vys cytoplasm (FIG. 3F). A control peptide FITC conjugate was notdetectable in the placenta (FIG. 3E). Localization of TPKTSVT (SEQ IDNO:5)-phage, TPKTSVT (SEQ ID NO:5)-GST, or TPKTSVT (SEQ ID NO:5)-FITC tocontrol organs, such as brain and pancreas, was not detected (data notshown). Together, these data show that the TPKTSVT. (SEQ ID NO:5)peptide targets the placenta, with the strongest homing noticed in thevys (FIG. 3B, FIG. 3D and FIG. 3F).

The TPKTSVT (SEQ ID NO:5) Peptide Binds to a Placental Transporter TheTPKTSVT (SEQ ID NO:5) peptide localizes to the vys (FIG. 3), which isthe tissue primarily responsible for materno-fetal transport in mice.This motif was tested to see if it would promote phage transport intothe embryo. Either TPKTSVT (SEQ ID NO:5)-phage or control phage wereinjected into pregnant mice and the recovery of phage from embryos wasdetermined. Specific accumulation of TPKTSVT (SEQ ID NO:5)-phage inembryos was observed to be up to 1,000-fold greater than that of controlphage (FIG. 4A). The TPKTSVT (SEQ ID NO:5) peptide was observed toapparently bind to a specific placental transporter. The materno-fetaltransfer of TPKTSVT (SEQ ID NO:5)-phage was blocked by an excess ofco-injected TPKTSVT (SEQ ID NO:5) peptide, but not the control GSTfusion peptide (FIG. 4A), suggesting transport by a specific receptorprotein as opposed to non-specific uptake.

Phage immunocytochemistry confirmed that the uptake of TPKTSVT (SEQ IDNO:5)-phage by the vys cells was not affected by the control GST fusionpeptide (FIG. 4A, FIG. 4B and FIG. 4C). FIG. 4B shows that TPKTSVT (SEQID NO:5)-phage administered alone are localized to the vys (arrows).FIG. 4C shows that in the presence of control-GST fusion peptides, theTPKTSVT (SEQ ID NO:5)-phage still localize to the vys (arrows). Incontrast, the addition of TPKTSVT (SEQ ID NO:5)-GST fusion peptideprevented internalization of TPKTSVT (SEQ ID NO:5)-phage into the vysepithelium (FIG. 4D). Together, these results show that the TPKTSVT (SEQID NO:5) peptide is actively transported through the placenta into theembryo by binding to a receptor located on endothelial cells in the vys.

The TPKTSVT (SEQ ID NO:5) Peptide Blocks Placental IgG Transport Thepattern of the TPKTSVT (SEQ ID NO:5) localization to the placenta (FIG.3) is reminiscent of that observed for IgG in that tissue (Parr andParr, 1985, J. Reprod. Immunol. 8: 153-171). Moreover, both IgG andTPKTSVT (SEQ ID NO:5) appear to undergo a receptor-mediated transportinto the embryo during pregnancy. It was hypothesized that IgG andTPKTSVT (SEQ ID NO:5) bind to a common receptor in the placenta. Theresults presented herein show that the TPKTSVT (SEQ ID NO:5) peptidecompetes for the placental transport of IgG.

Intravenous administration of a control GST-fusion peptide did notaffect the placental transfer of IgG to the vys (FIG. 5A, arrowheads).In contrast, co-administration of an equimolar dose of TPKTSVT (SEQ IDNO:5)-GST blocked translocation of IgG through the placenta (FIG. 5B).Although IgG localization to the labyrinthine blood vessels in theembryo-distal placental compartments was still detectable (FIG. 5B,asterisks), IgG staining in the vys epithelium was markedly decreased(FIG. 5B). The vys levels of IgG in the presence of TPKTSVT (SEQ IDNO:5)-GST were similar to those observed in β₂m-null mice (not shown),which are genetically deficient in IgG transcytosis (Israel et al.,1995, J. Immunol. 154:6246-6251; Zijlstra, et al., 1990, Nature344:742-746). Based on these data, it is proposed; that the TPKTSVT (SEQID NO:5) peptide selectively blocks transport through an immunoglobulinFc receptor that mediates the uptake of IgG by the yolk sac (FIG. 5C andFIG. 5D).

The TPKTSVT (SEQ ID NO:5) Receptor is Associated with the β₂m ProteinFcRn is a β₂m-associated Class I major histocompatibility complex(MHC-I) homologue. FcRN appears to regulate placental IgG transport(Ghetie and Ward, 2000, Ann. Rev. Immunol. 18:739-766; Simister andStory, 1997, J. Reprod. Immunol. 37:1-23). This is supported by theobservation that IgG species which are incapable of binding to FcRn arenot transported across the human placenta in an ex vivo model (Firan etal., 2001, Int. Immunol. 13:993-1002). In addition, FcRn expressionpatterns in the placenta resemble the pattern of placental IgGlocalization (Saji et al., 1999. Rev. Reprod. 4:81-89). A search of themouse protein database using BLAST software (NCBI;http://www.ncbi.nlm.nih.gov/BLAST/) revealed the similarity of theTPKTSVT (SEQ ID NO: 5) placental targeting peptide to the sequencePPKTTVT (amino acids 192-198 of the mouse MHC-I; Genbank accessionAAD43175). Moreover, the corresponding conserved human MHC-I sequence,PPKTHVT, is exposed on the surface of the MHC-I α₃ chain immediatelyadjacent to H-192 residue, a known β₂m contact site in the α₃ domain ofMHC-I homologues (Tysoe-Calnon et al., 1991, Biochem. J. 277:359-369).This makes the TPKTSVT (SEQ ID NO:5) motif an apparent mimeotope ofFcRn. Homing of the TPKTSVT (SEQ ID NO:5) peptide to the placenta,despite expression of FcRn in other tissues, may be due to eitherdifferential association of additional receptor subunits, or by alteredaccessibility of the receptor to the circulating ligand in the placenta.This is consistent with previous reports of FcRn interacting with IgG inthe placenta through a mechanism different from that in other tissues(Ghetie and Ward, Ann. Rev. Immunol. 18:739-766, 2000; Simister andStory, J. Reprod. Immunol. 37:1-23, 1997).

The TPKTSVT (SEQ ID NO: 5) motif was tested to determine if it targetsthe FcRn/β₂m receptor complex in the placenta. The β₂m-deficient mousestrain, in which FcRn is not functional (Isreal et al., 1995, J.Immunol. 154:6246-51; Zijlstra et al., 1990, Nature 344:742-746), wasused as a model system to test whether the TPKTSVT (SEQ ID NO:5) peptideis a ligand for the FcRn/β₂m receptor complex. Phage displaying theTPKTSVT (SEQ ID NO:5) peptide were intravenously injected into pregnantβ₂m-null mice and the recovery of phage from the placenta was assayed(FIG. 6). While the TPKTSVT (SEQ ID NO:5)-phage (FIG. 6A, white bars)homed to the wild-type (+/+) placenta relative to control phage (FIG.6A, black bars), no such homing was detectable in β₂m-deficient mice(FIG. 6A). Immunohistochemical analysis of phage accumulation in β₂mwild-type (FIG. 3B) and β₂m-null (FIG. 6B) placentas confirmed thatTPKTSVT (SEQ ID NO:5)-phage were not taken up by the vys epithelium inthe β₂m-deficient mice. In contrast, in vivo localization of a controlphage displaying a different placenta homing peptide, YIRPFTL (SEQ IDNO:9), which, unlike TPKTSVT (SEQ ID NO:5) peptide, homes to thevasculature of the labyrinthine placenta, rather than to the vys, wasnot affected in β₂m-null mice (FIG. 6C, arrowheads). Together, theseobservations strongly suggest that the TPKTSVT (SEQ ID NO:5) motiftargets the placenta by binding to FcRn/β₂m.

The TPKTSVT (SEQ ID NO: 5) Peptide Interferes with Mouse PregnancyBecause FcRn/β₂m regulates materno-fetal exchange, the aboveobservations suggested that TPKTSVT (SEQ ID NO:5) might interfere withplacental transport and embryonic development. TPKTSVT (SEQ ID NO:5)peptide was administered to pregnant mice in three differentforms—displayed on the phage capsid, fused to GST or fused to FITC. Thephage or fusion peptides were subcutaneously injected and theprogression of pregnancy was compared with mice injected with controlphage, control peptides, or saline. Multiple injections of the TPKTSVT(SEQ ID NO:5) peptide were administered, starting at mid-pregnancy (˜12days postconception, dpc) in doses non-toxic to the mother.

The effect of TPKTSVT (SEQ ID NO:5) peptide on fetal development isillustrated in FIG. 7A and FIG. 7B. TPKTSVT (SEQ ID NO:5) peptideinhibited the progression of pregnancy, as evidenced by diminishedweight gain in mice injected with the TPKTSVT (SEQ ID NO:5) phage or GSTfusion peptide compared to control phage or GST-peptide (FIG. 7A). Thecourse of pregnancy courses in mice injected with control peptides wereundistinguishable from those in saline-injected mice (data not shown).

Examination of embryos on the 20th day of pregnancy (the delivery dayfor the control) revealed that the TPKTSVT (SEQ ID NO:5) peptide has asevere effect on embryonic development. Growth-retarded, dead, orpartially resorbed embryos were observed in mice injected with eitherTPKTSVT (SEQ ID NO:5)-phage, TPKTSVT (SEQ ID NO:5)-GST, or TPKTSVT (SEQID NO:5)-FITC (FIG. 7B, embryo to right of figure). The normaldevelopment of a mouse embryo injected with control peptide is shown onthe left side of FIG. 7B. Administration of TPKTSVT (SEQ ID NO:5) fusionpeptides to pregnant mice resulted in 43% complete embryo resorption and21% dead or malformed conceptuses (FIG. 7B). The extent of embryoresorption and frequency of complete pregnancy abortion increased withprolonged TPKTSVT (SEQ ID NO:5) treatment (data not shown), suggestingthat the peptide effect is dose-dependent. Embryo death or morbiditywere not observed in any of the control groups.

Morphologic inspection of tissues from TPKTSVT (SEQ ID NO:5)peptide-injected mice revealed that, in contrast to controls, placentaswere edematous and grossly deformed. Histopathological examination ofthe placentas showed that the TPKTSVT (SEQ ID NO:5) treatment inducedmassive dilation of blood vessels and intraplacental bleeding, as wellas widespread hemorrhagic necrosis (FIG. 7D). Hematoxylin staining ofthe placental epithelium after seven days of peptide administrationshowed that most nuclei in the yolk sac and many in the labyrinthinecompartment were degraded (FIG. 7D). Also, massive fibrosis was evidentin the yolk sac cavity and in the labyrinthine portion of the placenta(FIG. 7D). In contrast, placentas from mice injected with controlpeptides (FIG. 7C) were indistinguishable from untreated placentas atcorresponding stages of pregnancy. The effect of TPKTSVT (SEQ ID NO:5)peptide on the reproductive system was strikingly specific, ashistological examination of control organs revealed no pathologicalchanges or necrosis, and no signs of peptide toxicity to the mother wereobserved (data not shown).

The teratogenicity observed with TPKTSVT (SEQ ID NO:5) is probably notcaused by the disruption of the FcRn/β₂M receptor function, as thefertility of mice is not significantly affected by β₂M deficiency(Zijlstra et al., Nature 344:742-46, 1990). Rather, embryotoxicity islikely secondary to placental thrombosis and ischemia. An alternativemechanism could be activation of complement and an immune responseagainst the targeting peptide itself.

These results have important implications for the development ofpregnancy-safe therapeutics, as it appears that a substance can causeembryotoxicity by merely homing to a placental cell surface markerwithout inactivating the receptor function. This creates the basis for ahigh throughput identification system based on placental receptors proneto teratogen binding. Systematic screening of potential teratogens forbinding to such receptors could be used to identify teratogenic activityand decrease the risk of teratogen induced birth defects.

Example 3 Identification of Mouse Adipose Targeting Peptides

Adipose Targeting Peptides

A similar protocol to that disclosed in Example 1 was used to isolatefat targeting peptides from a genetically obese mouse (Zhang et al.,Nature, 372:425-432, 1994; Pelleymounter et al., Science 269:540-543,1995). Phage that had been subjected to biopanning in obese mice werepost-cleared in a normal mouse. The fat-targeting peptides isolatedincluded TRNTGNI (SEQ ID NO:14), FDGQDRS (SEQ ID NO:15); WGPKRL (SEQ IDNO:16); WGESRL (SEQ ID NO:17); VMGSVTG (SEQ ID NO:18), KGGRAKD (SEQ IDNO:19), RGEVLWS (SEQ ID NO:20), TREVHRS (SEQ ID NO:13) and HGQGVRP (SEQID NO:21).

Homology searches identified several candidate proteins as theendogenous analogs of the fat targeting peptides, including stem cellgrowth factor (SCGF) (KGGRAKD, SEQ ID NO:19), attractin (mahogany)(RGEVLWS, SEQ ID NO:20), angiopoitin-related adipose factor (FIAF)(TREVHRS, SEQ ID NO:13), adipophilin (ADRP) (VMGSVTG, SEQ ID NO:18),Flt-1 or procollagen type XVII (TRNTGNI, SEQ ID NO:14) and fibrillin 2or transferrin-like protein p97 (HGQGVRP, SEQ ID NO:21)

Validation of Adipose Targeting Peptides

The fat homing peptides were validated by in vivo homing, as shown inFIG. 2. The fat homing clones selected were: FA—KGGRAKD (SEQ ID NO:19),FC—RGEVLWS (SEQ ID NO:20), FE—TREVHRS (SEQ ID NO:13) and FX—VMGSVTG (SEQID NO:18). As seen in FIG. 2, all of these clones exhibited someelevation of homing to adipose tissue, with clone FX showing severalorders of magnitude higher adipose localization than control fd-tetphage. Clone FX also exhibited substantially higher localization thanthe other selected fat homing clones. However, by analogy with theplacental homing peptides disclosed above, the skilled artisan willrealize that fat homing clones exhibiting lower levels of adipose tissuelocalization may still be of use for targeted delivery of therapeuticagents.

The skilled artisan will realize that targeting peptides selective forangiogenic vasculature in adipose tissue could be of use for weightreduction or for preventing weight gain. By attaching anti-angiogenic ortoxic moieties to an adipose targeting peptide, the blood vesselssupplying new fat tissue could be selectively inhibited, preventing thegrowth of new deposits of fat and potentially killing existing fatdeposits.

Example 4 CKGGRAKDC (SEQ ID NO: 22) Homes to White Fat in ob/ob Mice

Materials and Methods

Experimental Animals

C57BL/6 mice were purchased from Harlan Teklad. Leptin-deficient (ob/ob)(stock 000632) and leptin receptor-deficient (stock 000642) mice werepurchased from Jackson Laboratories (Bar Harbor, Me.). Anesthesia wasperformed with Avertin (0.015 ml/g) administered intraperitoneally(Arap, et al., 1998; Pasqualini & Rouslahti, 1996).

In Vivo Phage Library Screening

In vivo phage-display screening of the CX₇C library (C, cysteine; X, anyamino acid) (Pasqualini et al., 2000; Arap et al., Nature Med.8:121-127, 2002) for fat-homing peptides was performed (Pasqualini &Rouslahti 1996, Pasqualini et al., 2000). In each biopanning round, anadult ob/ob mouse was injected intravenously (tail vein) with 10¹⁰transducing units (Tu) of the library. Phage (˜300 TU/g in round 1increased to ˜10⁴ Tu/g in round 3) were recovered after 5 min ofcirculation by grinding subcutaneous white fat with a glass Douncehomogenizer, suspending the homogenate in 4° C. Dulbecco's ModifiedEagle's medium (DMEM) containing proteinase inhibitors (DMEM-prin: 1 mMPMSF, 20 μg/ml aprotinin, and 1 μg/ml leupeptin) and washing withDMEM-prin. The lipid phase was discarded during the washes and only thesolid-phase cellular material was used. Washed homogenates wereincubated with host bacteria (log phase E. coli K91kan; OD_(600 ˜)2).Bacterial cultures were plated onto Luria-Bertani agar plates containing40 μg/ml tetracycline and 100 μg/ml kanamycin, incubated overnight at37° C. and selected clones were bulk-amplified and used to precipitatephage for a subsequent round of biopanning. The sub-library amplifiedafter the third round of panning was enriched for fat-specific bindersusing a subtraction step. A lean C57BL/6 female was injected (tail vein)with 10⁹ TU of phage selected in round 3. After 5 min of circulation,the unbound phage were recovered from plasma and amplified for thefourth and final round of biopanning. In this protocol, phage that boundto tissues other than adipose were removed from the sub-library,increasing the selectivity of the recovered phage for binding to adiposetissue.

Peptide Localization in Tissues

Staining of formalin-fixed, paraffin-embedded mouse tissue sections wasperformed (Pasqualini & Rouslahti, 1996; Pasqualini et al., 2000). Forphage-peptide immunolocalization, 10¹⁰ TU of CKGGRAKDC (SEQ IDNO:22)-phage or a control insertless phage was injected intravenously.Phage immunohistochemistry was performed using a rabbit anti-fd phageantibody (Sigma Chemicals, St. Louis, Mo.) used at 1:1,000 dilution anda secondary horseradish peroxidase (HRP)-conjugated antibody. Apoptosiswas detected using standard TUNEL immunohistochemistry and anHRP-conjugated antibody. For in vivo peptide homing validation, stocksof 5-carboxyfluorescein (FITC)-conjugated CKGGRAKDC (SEQ ID NO:22) orCARAC (SEQ ID NO:12) were chemically synthesized, cyclized using theterminal cysteines and HPLC-purified to >90% purity by Anaspec (SanJose, Calif.). Lyophilized peptides were dissolved in DMSO to aconcentration of 20 mM. Ten μl of 1 mM peptide-FITC solution in PBS wasinjected 5 min prior to tissue extraction. For blood vessellocalization, 10 μl of 2 mg/ml of rhodamine-conjugated lectin-I(RL-1102, Vector Laboratories, Burlingame, Calif.) was co-injected. Allimmunohistochemistry and FITC immunofluorescence images were capturedusing an Olympus IX70 microscope and digital camera setup (Melville,N.Y.).

Anti-Obesity Therapy

Stocks of CKGGRAKDC (SEQ ID NO:22) fused to (KLAKLAK)₂ (SEQ ID NO:1);(KLAKLAK)₂ (SEQ ID NO:1) alone; CARAC (SEQ ID NO:12) fused to (KLAKLAK)₂(SEQ ID NO:1); and CKGGRAKDC (SEQ ID NO:22).peptide were chemicallysynthesized, cyclized using the terminal cysteines and HPLC-purifiedto >90% (Anaspec). Lyophilized peptides were dissolved in DMSO to aconcentration of 65 mM to make stock solutions. A total of 150 μl of0.65 mM peptide solution in PBS was subcutaneously injected daily in theback of C57BL/6 males, after body mass was measured each day. High-fatcafeteria diet for obesity induction (TD97366: 25.4% fat, 21.79%protein, 38.41% carbohydrate) was purchased from Harlan Teklad. Micewere pre-fed with TD97366 prior to the initiation of treatment withadipose targeting peptides to induce diet-related obesity. The high-fatdiet resulted in an average weight of 50 g before treatment.

Results

In vivo phage display (Pasqualini and Ruoslahti., Nature 380:364-366,1996; Kolonin et al., Curr. Opin. Chem. Biol. 5:308-313, 2001;Pasqualini et al., In Vivo Phage Display, In Phage Display: A LaboratoryManual, eds. Barbas et al., pp. 1-24. Cold Spring Harbor LaboratoryPress, New York, 2000) was used as described above to obtain a peptidetargeting the fat vasculature. A phage-display library was screened forpeptide motifs that home to the vasculature of subcutaneous white fat inmorbidly obese leptin-deficient (ob/ob) mice (Zhang et al. Nature372:425-432, 1994). This model provides a convenient source of adiposetissue. Four rounds of panning were followed by a fat-specific in vivosubtraction to restrict ligands to those binding to adipose-specificendothelial receptors. The DNA encoding the correspondingphage-displayed peptides was then sequenced to obtain the targetingpeptide amino acid sequences. Statistical analysis of selected motifsusing SAS software (version 8, SAS Institute) revealed that the motifCKGGRAKDC (SEQ ID NO:22) constituted 4.5% of all clones identified inthe screen. Intravenous administration of this clone into ob/ob miceshowed that CKGGRAKDC (SEQ ID NO:22)-phage accumulated in subcutaneousfat to a higher level than a control insertless phage (data not shown).

The tropism of CKGGRAKDC (SEQ ID NO:22)-phage for adipose tissue wasconfirmed by immunohistochemistry: CKGGRAKDC (SEQ ID NO:22)-phage showedmarked localization to the vasculature of subcutaneous and peritonealwhite fat (FIG. 8 a, arrows), whereas the control phage was undetectablein fat blood vessels (FIG. 8 b). To test whether targeting of theCKGGRAKDC (SEQ ID NO:22) motif to the fat vasculature would also occurwhen the peptide is outside of the context of the phage, the in vivodistribution of intravenously injected CKGGRAKDC (SEQ ID NO:22) peptidefused to fluorescent (FITC) was determined. Immunofluorescence insubcutaneous and peritoneal fat from peptide-injected ob/ob mice showedthat CKGGRAKDC (SEQ ID NO:22)-FITC localized to and was internalized bycells of white adipose vasculature (FIG. 8 c, arrows), whereas a controlCARAC (SEQ ID NO:12)-FITC conjugate was undetectable in adipose tissue(FIG. 8 d).

CKGGRAKDC (SEQ ID NO:22) Homes to White Fat in Wild-Type Mice

The mutation in leptin that leads to the extreme proliferation of whiteadipose tissue in mice (Zhang et al., 1994) is not frequentlyencountered in humans (Ozata et al., J. Clin. Endocrinol. Metab.84:3686-3695. 1999). Thus, this animal model may not be representativeof the typical pattern of obesity in humans. To exclude the possibilitythat CKGGRAKDC (SEQ ID NO:22) homing to fat is limited to ob/ob mice andto demonstrate the general applicability of adipose-targeting peptidesfor naturally-occurring obesity, the CKGGRAKDC (SEQ ID NO:22) peptidewas tested in wild-type mice.

FIG. 9 shows that the CKGGRAKDC (SEQ ID NO:22)-FITC fusion peptideintravenously injected into C57BL/6 (leptin +/+) mice specificallylocalized to blood vessels of subcutaneous and peritoneal white fat(FIG. 9A, FIG. 9B). A lectin-rhodamine peptide was used to visualizeblood vessel endothelium (arrows, FIG. 9B, FIG. 9D, FIG. 9F). TheCKGGRAKDC (SEQ ID NO:22)-FITC fusion peptide co-localized withlectin-rhodamine in adipose tissue (arrows, FIG. 9A and FIG. 9B). Nosuch co-localization was observed in control pancreatic tissue (FIG. 9Cand FIG. 9D) or other control organs (data not shown). The control CARAC(SEQ ID NO:12)-FITC peptide was not detectable in white fat vasculature(FIG. 9E and FIG. 9F). These in vivo localization data show that theadipose-targeting CKGGRAKDC (SEQ ID NO:22) peptide targets the whiteadipose vasculature in genetically normal obese mice as well as inleptin deficient mice, demonstrating the general applicability ofadipose targeting using such peptides. The uptake of CKGGRAKDC (SEQ IDNO:22)-FITC by the endothelium of fat tissue suggests that the motiftargets a receptor selectively expressed in the adipose vasculature thatcould provide a mechanism for directed delivery of therapeutic compoundsto fat.

Design and Use of Fat-Targeted Pro-Apoptotic Peptide

It was next determined whether proliferation of adipose tissue could becontrolled via targeted destruction of the fat vasculature. Thepro-apoptotic peptide KLAKLAKKLAKLAK (SEQ ID NO:1) (Ellerby et al.,Nature Med. 5:1032-38, 1999) (designated KLAKLAK)₂), which disruptsmitochondrial membranes to induce apoptosis, has been targeted toreceptors in tumor vasculature via a conjugated homing peptide (Ellerbyet al 1999, Arap, et al., Proc. Natl. Acad. Sci. U.S.A. 99:1527-1531,2002). The (KLAKLAK)₂ (SEQ ID NO:1) peptide was conjugated to the fattargeting CKGGRAKDC (SEQ ID NO:22) peptide for targeted delivery to fatvasculature in adipose tissue. The D enantiomer of (KLAKLAK)₂ (SEQ IDNO:1), which is resistant to proteolysis but still exhibitspro-apoptotic activity, was conjugated to the CKGGRAKDC (SEQ ID NO:22)peptide via a glycinylglycine bridge. The conjugated fat-targeting,pro-apoptotic peptide was administered to mice and the effect on adiposetissue was monitored.

A non-genetic mouse obesity model was initially used. A cohort ofC57BL/6 (wild-type) mice, in which obesity had been induced by ahigh-fat cafeteria diet, were subcutaneously injected with CKGGRAKDC(SEQ ID NO:22)-(KLAKLAK)₂ (SEQ ID NO:1) peptide and weighed daily over aperiod of two weeks. Cafeteria dieting continued throughout theexperiment. As shown in FIG. 10A, injections of CKGGRAKDC (SEQ ID NO:22)conjugated to (KLAKLAK)₂ (SEQ ID NO:1) prevented obesity development andsurprisingly caused a rapid decrease in body mass of up to 20%. Incontrast, obese mice injected with two negative controls (an equimolaramount of either unconjugated CKGGRAKDC (SEQ ID NO:22) and (KLAKLAK)₂(SEQ ID NO:1) or a control CARAC (SEQ ID NO:12)-(KLAKLAK)₂ (SEQ ID NO:1)conjugate) did not show a significant body mass decrease and continuedto increase in weight (FIG. 10A).

The effectiveness of the CKGGRAKDC (SEQ ID NO:22)-(KLAKLAK)₂ (SEQ IDNO:1) conjugate was also examined in wild-type mice fed on a regulardiet (FIG. 10B). C57BL/6 mice that had developed a considerable amountof subcutaneous and peritoneal fat due to old age were subcutaneouslyinjected with the CKGGRAKDC (SEQ ID NO:22)-(KLAKLAK)₂ (SEQ ID NO:1)conjugate or control peptides over a period of one month. As in thediet-induced obesity model, targeting of (KLAKLAK)₂ (SEQ ID NO:1) to fatby conjugation with CKGGRAKDC (SEQ ID NO:22) resulted in greater than35% reduction in body mass at a rate of 10% per week (FIG. 10B). Notoxicity of the conjugated peptide was detected under these conditions(data not shown). In fact, the CKGGRAKDC (SEQ ID NO:22)-(KLAKIAK)₂ (SEQID NO:1) treated mice became more active and agile following body massreduction and appeared healthier than prior to treatment (data notshown). The control untargeted (KLAKLAK)₂ (SEQ ID NO:1) treatmentsresulted in only a slight body mass reduction (FIG. 10B), possibly dueto low levels of nonspecific toxicity. The control mice did not exhibitthe increased activity and/or agility seen in treated mice (data notshown).

Fat Resorption with CKGGRAKDC (SEQ ID NO:22)-(KLAKLAK)₂ (SEQ ID NO:1) isMediated by Apoptosis

In both diet-induced and age-related obesity, the effect of CKGGRAKDC(SEQ ID NO:22)-(KLAKLAK)₂ (SEQ ID NO:1) treatment on body mass was dueto fat resorption, which was visually apparent by the end of treatment(FIG. 11). Wild-type mice were fed on a high fat cafeteria diet (FIG.11A). Alternatively, wild-type fed on a regular diet became obese as aconsequence of old age (FIG. 11B, FIG. 11C, FIG. 11D). Mice were treatedwith CKGGRAKDC (SEQ ID NO:22) conjugated to (KLAKLAK)₂ (SEQ ID NO:1)(left side of FIG. 11), with CARAC (SEQ ID NO:12) conjugated to(KLAKLAK)₂ (SEQ ID NO:1) (middle of figure), or with unconjugatedCKGGRAKDC (SEQ ID NO:22) and (KLAKLAK)₂ (right side of FIG. 11).

Gross inspection of mouse organs revealed that both subcutaneous (FIG.11B) and visceral (FIG. 11C) fat exhibited marked resorption upontreatment with CKGGRAKDC (SEQ ID NO:22) conjugated to (KLAKLAK)₂ (SEQ IDNO:1) (right side of FIG. 11). Quantification of fat resorption afterthree weeks of treatment by weighing a specific fat depot (epididymalfat, FIG. 11D) showed a greater than 3-fold reduction in fat masscompared with controls (FIG. 11D, left side of figure compared to middleand right side).

Histopathological analysis of tissues from mice treated with CKGGRAKDC(SEQ ID NO:22) conjugated to (KLAKLAK)₂ (SEQ ID NO:1) showed vascularapoptosis (FIG. 12A, arrows) and resulting fat necrosis with lymphocyteinfiltration (FIG. 12C, arrows) in adipose tissue following treatment.In contrast, mice treated with a control fusion peptide comprising CARAC(SEQ ID NO:12) conjugated to (KLAKLAK)₂ (SEQ ID NO:1) showed no vascularapoptosis or fat necrosis (FIG. 12D). No abnormalities in other organstreated with CKGGRAKDC (SEQ ID NO:22) conjugated to (KLAKLAK)₂ (SEQ IDNO:1) (data not shown).

Injection of CKGGRAKDC (SEQ ID NO:22) conjugated to (KLAKLAK)₂ (SEQ IDNO:1) into genetically obese mice, but not into normal obese mice, wasoccasionally observed to result in mortality within a few days ofinjection. It is not clear what the mechanism might be for inducingdeath in genetically obese mice, although development of pulmonary orcardiac fat embolism or rapid drop of serum calcium due tosaponification by released lipids are possibilities. However, theseresults suggest that treatment of grossly obese subjects might result insufficient adipose cell death and necrosis to adversely affect thehealth of the subject, indicating that lower dosages and/or use of atime release formulation of the adipose targeting conjugate may bepreferred in cases of excessive obesity.

Adipose Receptor Protein for CKGGRAKDC (SEQ ID NO:22)

A band of approximately 35,000 Daltons (35 kDa) was isolated from mouseadipose tissue extract that bound to CKGGRAKCDC (SEQ ID NO:22)conjugated to (KLAKLAK)₂ (SEQ ID NO:1). There was much less binding ofthe 35 kDa fraction to the control peptide CARAC (SEQ ID NO:12)conjugated to (KLAKAK)₂ (SEQ ID NO:1) (data not shown). The 35 kDa bandwas analyzed by mass spectrometry, which identified three proteinspresent in the sample.

The three proteins included predominately a B cell receptor associatedprotein (prohibitin), apolipoprotein E, and the voltage dependent anionchannel (VDAC). Further studies were performed by immunoprecipitation,using either CKGGRAKDC (SEQ ID NO:22) or CARAC (SEQ ID NO:12) conjugatedto (KLAKAK)₂ (SEQ ID NO:1) and precipitating with commercially availableantibodies.

SDS-polyacrylamide gel electrophoresis of the immunoprecipitated proteinshowed that only the prohibitin receptor protein complex wassubstantially enriched by binding to CKGGRAKDC (SEQ ID NO:22) (data notshown), with over a ten-fold enrichment in the CKGGRAKDC (SEQ ID NO:22)precipitated fraction compared to the CARAC (SEQ ID NO:12) precipitatedfraction (data not shown). The CARAC (SEQ ID NO:12)-(KLAKAK)₂ (SEQ IDNO:1) fusion peptide exhibited low levels of non-specific binding to allthree proteins (VDAC, prohibitin and apolipoprotein E). It is unknownwhether those proteins bound to the CARAC (SEQ ID NO:12) moiety or to(KLAKAK)₂ (SEQ ID NO:1).

It is concluded that the adipose tissue endothelial receptor forCKGGRAKDC (SEQ ID NO:22) is prohibitin (Genbank Accession No.NM_(—)008831). Probitin is expressed in mitochrondria of various celltypes and in the cell membrane of B lymphocytes. Immunohistochemicalanalysis shows that prohibitin is expressed in blood vessels of adiposetissues but not of other organs (data not shown). Based on theseresults, it is concluded that pro-apoptosis agents conjugated totargeting peptides that bind to a prohibitin receptor protein complexare effective to induce adipose cell death and weight loss in obesesubjects. The skilled artisan will realize that other prohibitin-bindingtargeting peptides, antibodies, etc. may be used within the scope of theclaimed methods and compositions to control weight and/or to induceweight loss. Further, other known cytocidal, cytotoxic and/or cytostaticagents may be used in place of (KLAKAK)₂ (SEQ ID NO:1) to control weightor induce weight loss within the scope of the claimed subject matter.

Example 5 Screening an Alpha-Spleen Antibody Library In Vivo by BRASIL

The following Examples are illustrative of general techniques that maybe of use in various embodiments of the claimed invention. As part ofthe reticulo-endothelial system, biopanning against spleen tissue iscomplicated by the high background of non-specific phage localization tospleen. The decreased background observed in biopanning with the BRASILmethod is advantageous for identifying targeting peptides againsttissues such as spleen.

This example demonstrates an illustrative embodiment of the BRASILmethod. A phage library based on immunoglobulins derived against thetarget organ (mouse spleen) was developed and then subjected to in vivobiopanning. To construct the immunoglobulin library, mouse spleen wasinjected into a chicken. After boosting, the chicken spleen wascollected and immunoglobulin variable domain sequences were obtained byPCR™ amplification of chicken spleen mRNA. The amplified immunoglobulinvariable sequences were inserted into a phage display library(α-library) that was then used for in vivo biopanning against mousespleen. Thus, the spleen targeting peptide sequences obtained from phagelocalized to mouse spleen in vivo were derived from antibody fragmentsproduced in the chicken in response to mouse spleen antigens. Thesuccess of this example further shows the broad utility of the BRASILmethod. The skilled artisan will realize that the present invention isnot limited to the embodiments disclosed herein and that many furtherdevelopments of the BRASIL methodology are included in the scope of thepresent invention.

Materials and Methods

Library Construction

A white leghorn chicken was immunized with spleen homogenate (about 150mg per injection) from a perfused (10 ml MEM) Balb/c mouse. The chickenreceived spleen homogenate boosters at 4 weeks and 8 weeks after theinitial immunization. Immune response to mouse spleen by FACS analysisshowed that the chicken immune serum contained antibodies against amouse cell-line (TRAMP-C1). The chicken was sacrificed and its spleenwas removed to TRI Reagent (Molecular Research Center, Inc., Cincinnati,Ohio) 12 weeks after the first immunization.

Total RNA was prepared from the chicken spleen using the manufacturer'sprotocol for the TRI reagent. cDNA was prepared from the total RNA usingoligo. (dT)-primers and Superscript enzyme (Life Technologies,Gaithersburg, Md.). cDNAs encoding chicken spleen immunoglobulinvariable regions were amplified by CHybVH and ChybIgB (V_(heavy)) or byCSCVK and CHHybL-B (V_(kappa)) primers according to standard techniques.Light chain variable regions and constant regions were PCR™ amplifiedtogether using CSC-F and lead-B primers and V_(kappa) and C_(kappa)templates. Heavy chain variable regions and constant regions were PCR™amplified together using dp-seq and lead-F primers and V_(heavy) andC_(heavy) templates. Heavy- and light-chain fragments were PCR™amplified together with CSC-F and dp-Ex primers. PCR primers werepurchased from Genosys (The Woodlands, IX) or GenBase (St. Lucia,Queensland, Australia), using primer sequences listed in the Cold SpringHarbor laboratory course manual, “Phage Display of CombinatorialAntibody Libraries” (Barbas et al., 2000), the relevant text of which isincorporated herein by reference.

After digestion with Sfi I, the amplification products were ligated toSfiI-digested pComb3x for insertion into the phage library. LigatedpComb3-123 plasmid was electroporated into ER2537—E. coli and phageproduction was started with subsequent VCM13 (helper phage) infection.The resulting library size was about 5×10⁶ cfu.

In Vivo Screening of α-Spleen Library Using BRASIL

Four rounds of in vivo screening in mice were performed using thechicken α-spleen library. About 0.8 to 2.0×10¹⁰ TU were injected into aBalb/c mouse. The library was allowed to circulate for 5 minutes. Aftersacrifice, the mouse spleen was recovered and a single cell suspensionwas prepared by pressing the spleen through a 70 μm cell strainer nylonmesh. The single cell suspension was centrifuged over oil (9:1 dibutylphtalate: cyclohexane) using the BRASIL technique and 200 μl of logphase ER2537 E. coli were infected with the pellet. Amplified phagerecovered from the mouse spleen was used for the subsequent round ofscreening. No obvious enrichment in the screening rounds was seen in thenumber of phage homing to spleen and brain compared with theconventional biopanning method, using a piece of spleen obtained priorto BRASIL.

Phage localized in mouse spleen from the fourth round of screening ofthe chicken Fab inserts were PCR™ amplified and the PCR product wasdigested with Bst I. Half of the clones out of 90 analyzed produced asimilar restriction pattern. Of those, 20 clones were sequenced fromwhich only two had an identical restriction pattern. Four of theantibody based phage clones (numbers 2, 6, 10 and 12) were subjected tofurther analysis using binding and localization assays.

Testing the Clones In Vitro Using BRASIL

A singe cell suspension was prepared from two mouse spleens. Thesuspension was divided into five tubes and incubated on ice with 3×10⁹TU of Fab clones #2, #6, #10, #12 and 2×10⁹ TU tet-phage. Phage bound tomouse spleen cells were recovered by BRASIL. 200 μl of log phase ER2537E. coli was infected with the pellet and serial dilutions were plated onLB/carbenicillin and LB/tetracycline plates for assessment of phagebinding. Fd-tet was used as an internal control to normalize all thephage homing experiments.

Testing Clones In Vivo with BRASIL

Phage (3×10⁹) of Fab clones #2, #6, #10, #12 and 2×10⁹ TU tet-phage wereinjected into the tail veins of Balb/c mice and allowed to circulate for5 minutes. The spleens were recovered and single cell suspensions wereprepared on ice from whole spleens. Cell bound phage were recovered byBRASIL. 200 μl of log phase ER2537 E. coli was infected with the pelletand serial dilutions were plated on LB/carbenicillin and LB/tetracyclineplates for assessment of the phage recovery.

Testing Clone #10 Versus, Control Phage NPC-37T In Vivo with BRASIL

Phage (3×10⁹ TU) of Fab clone #10 and NPC-3TT (control Fab phage) and1×10⁹ TU of control Fd-tet-phage were injected to mice (2 mice forNPC-3TT, 2 mice for clone #10) and allowed to circulate for 5 minutes.Spleens were recovered and single cell suspensions were prepared on ice.Cell-bound phage were recovered by BRASIL. 200 μl of log phase ER2537 E.coli was infected with the pellet and serial dilutions were plated onLB/carbenicillin and LB/tetracycline plates. The NPC-3TT phage is ahuman anti-tetanus toxin Fab fragment displaying phage.

Homing of Fab Clone #10 to Spleen Versus Bone Marrow

Phage (3×10⁹ TU) of Fab clone #10 and NPC-3tt control and 1×10⁹ TU ofFd-tet control phage were injected into mice (2 mice for NPC-3TT, 2 micefor clone #10) and allowed to circulate for 5 minutes. The spleens wererecovered and single cell suspensions were prepared. Bone marrow wasrecovered from the same mice (both femurs) as a control for organspecific homing. Cell-bound phage were recovered by BRASIL.

Fab-Fragment Production

The plasmid pComb3 containing the chicken Fab inserts was electroporatedinto ER2537 bacteria. Serial dilutions were plated onto LB/carbenicillinplates and incubated overnight at 37° C. Fab production culture (insuper broth with 100 μg/ml carbenicillin) was started from a singleplated colony. Fab production was induced with 1 mM IPTG for 7 hours at30° C. The Fab fragment was purified from the periplasmic fraction SN2by affinity purification after determination of the Fab concentration inbacteria supernatant, periplasmic fractions SN1 and SN2 and in thebacteria lysate by ELISA. An α-Fab-Protein G-column was coupled (2mg/ml) with dimethylpimelimidate (DMP) using standard protocols (Harlowand Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, New York, N.Y., 1988).

For purifying Fab fragments the following method was used. The SN2fraction was loaded into a 1 ml HiTrap-protein G-α-Fab-column (AmershamPharmacia Biotech, Piscataway, N.J.) either over 2 hours (if using lowerthan 50 ml volume with superloop) or overnight (with more than 50 mlvolume using a peristaltic pump). The column was washed with 10-20 ml ofPBS (phosphate buffered saline). The Fab fragments were eluted with 10ml of 20 mM glycine buffer, pH 2.2, 150 mM NaCl and 1 ml fractions werecollected. Fractions are neutralized with 1 M Tris immediately afterelution. Protein concentrations were quantified by A₂₈₀.

Intravascular Staining

To determine in vivo distribution of the recovered Fab fragments, 50 to60 μg of Fab fragment (Fab#10, NPC3-tt or R#16) was injected into thetail vein of a Balb/c mouse and allowed to circulate for 8 minutes. 50μg of L. esculentum lectin-FITC was injected into the mouse and themouse tissues were fixed by perfusion with 25 to 30 ml of 4%paraformaldehyde/PBS after 2 minutes of lectin circulation. Tissues wereremoved and post-fixed in 4% paraformaldehyde for 1 hour. Fixed tissueswere incubated in 30% sucrose/PBS overnight at 4° C., changing thesolution at least twice. The tissues were embedded in the freezing mediaand frozen on dry ice.

Fixed tissue sections were stained for Fab as follows. Frozen tissuesections (55 μm) were cut on a microtome and washed 3× with PBS. Thethin sections were blocked with PBS/0.3% TritonX-100/5% goat serum for 1hr at room temperature. Sections were incubated overnight at roomtemperature with 1:400 Cy3 conjugated α-human anti-Fab antibody. Theconjugated sections were washed 6× with PBS/0.3% Triton X-100, 3× withPBS, and fixed with 4% paraformaldehyde for 15 minutes. After fixationthe sections were washed again 2× with PBS and 2× with distilled water,then mounted on slides using Vector Shield.

Results

The in vitro localization to mouse spleen cells of phage clonesexpressing chicken Fab fragments was examined by BRASIL. As shown inFIG. 13, the Fab phage clones isolated by BRASIL showed differentialbinding to mouse spleen cells compared to Fd-tet insertless controlphage. Clone #6 showed the lowest degree of binding, similar to thecontrol phage NPC-3TT, which contained a Fab fragment but was notisolated from mouse spleen. Clones #2, #10 and #12 all showed selectivebinding to mouse spleen cells compared to the Fd-tet control, with atleast a two-fold increased binding observed for clones #2 and #10 (FIG.13). The amino acid sequences determined for the clone inserts were:

Clone #2: (SEQ ID NO:23)CQPAMAAVTLDESGGGLQTPGGALSLVCKASGFTFNSYPMGWVRQAPGKGLEWVAVISSSGTTWYAPAVKGRATISRDNGQSTVRLQLSNLRAED Clone #6: (SEQ ID NO:24)CQPAMAAVTLDESGGGLQTPGGTLSLVCKASGISIGYGMNWVRQAPGKGLEYVASISGDGNFAHYGAPVKGRATISRDDGQNTVTLQLNNLR Clone #10: (SEQ ID NO:25)CQPAMAAVTLDESGGGLQTPGGTLSLVCKGSGFIFSRYDMAWVRQAPGKGLEWVAGIDDGGGYTTLYAPAVKGRATITSRDNGQSTVRLQLNNLR Clone #12: (SEQ ID NO:26)ANQPWPPLTLDESGGGLQTPGGALSLVCKASGFTMSSYDMFWVRQAPGKGLEFVAGISSSGSSTEYGAAVKGRATISRDNGQSTVRLQLNNLRAED

A direct comparison was made of in vitro phage binding for the Fabclones compared to NPC-3TT. As shown in FIG. 14, clones #2 and #10exhibited the highest levels of binding to mouse spleen cells in vitro.Clones #6 and #12 showed levels of binding to mouse spleen that wereonly slightly higher than the binding of phage NPC-3TT.

The preferential binding of the chicken Fab phage clones was confirmedby in vivo studies using BRASIL. As shown in FIG. 15, selectivelocalization to mouse spleen was even more dramatic in vivo, with Fabclones #2, #6 and #10 showing many-fold increased binding to spleencompared to Fd-tet phage. In contrast, Fab clone #12 did not exhibitsignificantly elevated binding to mouse spleen compared to Fd-tet phage.These results show that in vitro results obtained with spleen targetingphage are confirmed in vivo.

Fab clone #10 was selected for additional characterization by in vivolocalization to mouse spleen. The results, shown in FIG. 16, confirmthat Fab clone #10 exhibited 3 to 10 fold enrichment in spleen comparedto Fd-tet. This effect was not due to general Fab binding, since the Fabcontrol phage NPC-3TT did not exhibit selective localization in spleencompared to Fd-tet insertless phage.

Binding of Fab clone #10 was organ specific, as demonstrated in FIG. 17.Phage from Fab clone #10 and NPC-3TT control were recovered from spleenand bone marrow tissue from the same injected mice. It can be seen inFIG. 17 that Fab clone #10 exhibited selective localization to spleenbut not to bone marrow tissue. The control phage did not exhibitselective localization to bone marrow (FIG. 17) or spleen (not shown).

These results show that Fab phage clone #10 selectively targets mousespleen tissue for binding both in vitro and in vivo. These results werefurther validated by vascular staining for in vivo phage distribution.Control phage used for this study were clones NPC-3TT (Fab fragment) andclone R#1.6 (isolated from angiogenic retina screening).

Fab clone #10 was observed to bind to mouse spleen tissue in vivo byfluorescent staining (not shown). The control phage NPC-3TT and R#16 didnot stain spleen tissue under identical conditions (not shown). Theclone #10 and NPC-3TT phage were observed to intensively stain kidneysof injected animals, perhaps due to glomerular filtration (not shown).Other control organs (lung, brain, liver, heart and skeletal muscle) didnot show staining with clone #10 (not shown).

These results demonstrate that spleen targeting phage peptides can beidentified by the BRASIL method. They further show the feasibility ofthe phage display technique using antibody fragments against a targetorgan, tissue or cell type to obtain a starting phage library. Theability to obtain targeting peptides against spleen, a tissue that hasproven refractory to biopanning using standard phage display protocolsbecause of the high non-specific background, shows the advantages of theBRASIL method.

Example 6 Identification of Receptor/Ligand Pairs: Targeting PeptidesAgainst Integrin Receptors

Certain embodiments of the present invention concern the identificationof receptor/ligand pairs for various applications. Targeting peptidesselective for organs, tissues or cell types bind to receptors (asdefined above), normally located on the lumenal surface of blood vesselswithin the target. In certain embodiments, targeting peptides may beused to identify or characterize such receptors, either directly orindirectly. In addition to their use as targets for delivery of genetherapy vectors, other therapeutic agents or imaging agents for in vivoimaging, such naturally occurring receptors are of use as potentialtargets for development of new therapeutic agents directed against thereceptor itself, for development of vaccines directed against thereceptor, and for understanding the molecular mechanisms underlyingvarious disease states. Naturally, the targeting peptides themselves mayserve as the basis for new therapeutic agents directed against thereceptors.

Targeting peptides may frequently act as mimeotopes of endogenousligands that bind to the targeted receptor. In other embodiments, theendogenous ligands may be identified and characterized using thedisclosed methods. Such ligands are also of potential use as targets fordevelopment of new therapeutic agents, etc.

The present example illustrates one embodiment related to identificationof receptor/ligand pairs, in this case, integrin receptors. Non-limitingexamples of applications of targeting peptides directed againstintegrins include regulation of cell proliferation and chemotaxis,pro-apoptosis and anti-angiogenesis. In this embodiment, purifiedintegrins attached to a solid substrate were used to screen phagedisplay libraries to identify targeting peptides directed againstintegrins.

Background

Integrin function is regulated by cytokines and other soluble factors ina variety of biological systems. Most commonly, exposure to such factorsleads to conformational alterations that result in changes in theactivation state of the receptors (i.e., increased or decreased affinityfor a given ligand and/or receptor clustering in the plasma membrane).Changes in integrin-dependent adhesion ultimately activate variouscomplex signal transduction pathways. At the molecular level, theinduced co-localization of cytoskeleton proteins with integrincytoplasmic domains controls signal transduction.

Cytoplasmic domains are key regulators of integrin function (reviewed inHynes, Cell 69:11-25, 1992; Ruoslahti, Ann. Rev. Cell Dev. Biol.12:697-715, 1996). Individual α and β subunit cytoplasmic domains arehighly conserved among different species (Hemler et al, In: Integrins:The Biological Problems, ed. Takada, CRC Press, Inc. Boca Raton, Fla.,pp. 1-35, 1994). Although the cytoplasmic domains of various β subunitsshare similar primary structures, they differ in certain functionalcharacteristics. Experiments with chimeric integrins have shown that thecytoplasmic domains of β chains are responsible for regulating receptordistribution and recruitment to focal adhesion sites (Pasqualini andHemler, J. Cell. Biol. 125:447-460, 1994). Thus, certain cytoplasmicdomains are critical for integrin-mediated signaling into the cell(outside-in signaling) and activation of integrin-ligand bindingactivity (inside-out signaling) (Hemler et al., 1994).

The integrins αvβ3 and αvβ5 are selectively expressed in angiogenicvasculature but not in normal vasculature (Brooks et al., 1994a, 1994b;Pasqualini et al., 1997; Arap et al., 1998). Moreover, αv integrinantagonists have been shown to block the growth of neovessels (Brooks etal., 1994a, 1994b, 1995; Hammes et al., 1996). In these experiments,endothelial cell apoptosis was identified as the mechanism for theinhibition of angiogenesis (Brooks et al., 1994a, 1994b, 1995).Angiogenesis initiated by bFGF can be inhibited by an anti-αvβ3 blockingantibody, whereas VEGF-mediated angiogenesis can be prevented by ablocking antibody against αvβ5. The integrins αvβ3 and αvβ5 have beenreported to be preferentially displayed in different types of ocularneovascular disease (Friedlander et al., 1995, 1996). Thus, distinctcytokine-induced pathways that lead to angiogenesis seem to depend onspecific αv integrins.

The search for αv integrin-associated molecules has been hampered bytechnical difficulties. First, the physical associations involved arelikely to rely on an assembly of multimeric ligands that no longeroccurs when cells are not intact. Second, their association to integrinsis usually of low affinity. Finally, changes in the conformation andphosphorylation states of the associating proteins may add a furtherlevel of complexity in these transiently modulated interactions. Becauseof these problems, only a limited number of proteins that bind tointegrin cytoplasmic domains have been identified. These proteins, suchas paxillin and ICAP-1, mainly associate with the β1 chain (Shattil andGinsberg, 1997). Cytohesin-1 and filamin associate with the cytoplasmicdomain of β2.

The disclosed methods have several advantages over previous approaches:(i) the ability to characterize the intracellular molecules thatdirectly or indirectly interact with integrin cytoplasmic domains; (ii)the development of antibodies against molecules that bind to integrincytoplasmic domains in very low amounts; and (iii) the phage displaylibrary screenings will lead to the identification of peptides thatmimic cytoplasmic-domain binding proteins.

Methods

Two Dimensional Cell Culture

Three human endothelial cell lines that express β3 and β5 integrins wereused: KS1767 cells (Herndier et al., 1996), HUVECs (ATCC), and BCE cells(Solowska et al., 1991). Sterile glass coverslips covered with differentproteins (i.e. vitronectin, fibronectin, collagen, or laminin) were usedas substrates. After cells attached and spread, the monolayers wererendered quiescent by a 12-hour incubation in medium containing 0.05%fetal calf serum. Peptides were introduced into the cells using thepenetratin membrane-permeable tag (see below). The cells were platedonto ECM proteins for adhesion and spreading. The monolayer wasstimulated for 6 hours with each of the growth factors involved inav-mediated angiogenesis, including bFGF, TNFα, VEGF, and TGF™.Untreated cells were the negative controls.

Three-Dimensional Cell Culture:

150 μl of Matrigel were added per well of 24-well tissue culture platesand allowed to gel at 37° C. for 10 min. HUVECs starved for 24 h in M199medium supplemented with 2% FCS before being trypsinized were used. 10⁴cells were gently added to each of the triplicate wells and allowed toadhere to the gel coating for 30 min at 37° C. Then, medium was replacedwith peptides in complete medium. The plates were monitored andphotographed after 24 h with an inverted microscope (Canon).

Chemotaxis Assay:

Cell migration assays were performed as follows: 48-well microchemotaxischambers were used. Polyvinylpyrrolidone-free polycarbonate filters(Nucleopore, Cambridge, Mass.) with 8-μm pores were coated with 1%gelatin for 10 min at room temperature and equilibrated in M199 mediumsupplemented with 2% FCS. Peptides were placed in the lower compartmentof a Boyden chamber in M199 supplemented with 2% FCS, 20 ng/ml VEGF-A(R&D System), and 1 U/ml heparin. Overnight-starved subconfluentcultures were quickly trypsinized, and resuspended in M199 containing 2%FCS at a final concentration of 2×10⁶ cells/ml. After the filter wasplaced between lower and upper chambers, 50 μl of the cell suspensionwas seeded in the upper compartment. Cells were allowed to migrate for 5h at 37° C. in a humidified atmosphere with 5% CO₂. The filter was thenremoved, and cells on the upper side were scraped with a rubberpoliceman. Migrated cells were fixed in methanol and stained with Giemsasolution (Diff-Quick, Baxter Diagnostics, Rome, Italy). Five randomhigh-power fields (magnitude 40×) were counted in each well.

Proliferation Assay:

Cell proliferation was measured as described (Pasqualini and Hemler,1994). Briefly, 4×10⁴ HUVECs were incubated in 24-wells plates. Thecells were starved for 24 h, and then the medium was removed andreplaced in the presence of VEGF and 15 μM of each peptide and incubatedfor 18 h. Then, 50 μl of media containing [³H]thymidine (1 μCi/ml) wasadded to the wells, and after 6 additional hours of incubation at 37°C., the medium was removed and the cells were fixed in 10% TCA for 30min at 4° C., washed with ethanol, and solubilized in 0.5 N NaOH.Radioactivity was counted by liquid scintillation with an LS 6000SCBeckman scintillation counter. Each experiment was performed three timeswith triplicates, and the results are expressed as the mean±SD.

Apoptosis Assay (Propidium Iodide Staining Subdiploid Population)

Approximately 1×10⁶ cells were harvested in complete media and 15 μM ofpeptide added for 4, 8, or 12 h. The cells were then washed in PBS andresuspended in 0.5 ml propidium iodide solution (50 μg/ml PI, 0.1%Triton X-100, 0.1% sodium citrate). After a 24-h incubation at 4° C.,cells were counted with a XL Coulter (Coulter Corporation) with a 488-nmlaser; 12,000 cells were counted for each histogram, and cell cycledistributions were analyzed with Multicycle program.

After microinjection or penetratin-mediated internalization of thepeptides and appropriate controls, cell apoptosis was monitored usingthe ApopTag kit. Experiments were performed in the presence of caspaseinhibitors and antibodies against specific caspases.

Cytokine- and Tumor-Induced Angiogenesis Assays

Angiogenic factors and tumor cells implanted into CAM stimulate growthof new capillaries. Angiogenesis was induced in CAMs from 10-day chickenembryos by VEGF or bFGF filters implanted in regions that werepreviously avascular. Different treatments (penetratin peptides andcontrols) were applied topically, and after 3 days, the filters andsurrounding CAMs were resected and fixed in formalin. The number ofblood vessels entering the disk was quantified within the focal plane ofthe CAM with a stereomicroscope. The mean number of vessels and standarderrors from 8 CAMs in each group were compared.

Phosphorylation and Panning of Phosphorylated Phage Libraries

Phosphorylation of peptide libraries with src family protein kinases(Fyn, c-Src, Lyn, and Syc) and serine/threonine kinases such as a MAPkinase were performed as described previously (Schmitz et al., 1996;Dente et al., 1997; Gram et al., 1997). Briefly, phage particles werecollected from culture supernatants by double precipitation with 20%polyethylene glycol 8000 in 2.5 M NaCl. Particles were dissolved at 10¹²particles/ml. Purified phage (10 μl) were incubated for 3 hours at roomtemperature with different concentrations (35 to 3,500 units) of proteinkinases in a reaction buffer volume of 50 μl. The reaction mixtures weretransferred to tubes containing 10 μg of agarose-conjugated anti-P-Tyr,anti-P-Ser, or anti-P-Thr monoclonal antibodies to select phagedisplaying phosphorylated peptides. Bound phage were eluted by washingthe column with 0.3 ml of elution buffer (0.1 M NaCl/glycine/1 mg/mlBSA, pH 2.35). The eluates were neutralized with 2 M Tris-base andincubated with 2 ml of a mid-log bacteria culture. Aliquots of 20 μlwere removed for plating, and phage were harvested as described. Thephosphorylation-selection step was repeated. Phosphorylated peptidesbinding to 133 and 135 cytoplasmic domains were analyzed as described inthe previous section.

Matrix-assisted laser desorption time-of-flight (MALDI-TOF) massspectrometry was used to map in vitro phosphorylation sites on the β3and β5 cytoplasmic domains and cytoplasmic domain-binding peptides. Thefusion proteins or peptides were phosphorylated in vitro as describedand purified by RP-HPLC or RP microtip columns. Phosphorylated peptideswere identified by three methods: (1) 80-Da mass shifts after kinasereactions; (2) loss of 80 Da after phosphatase treatment; or (3) loss of80 Da or 98 Da in reflector vs. linear mode for tyrosine phosphorylatedor serine, threonine phosphorylated peptides, respectively. Whereneeded, peptides were purified by RP-HPLC and subjected tocarboxypeptidase and aminopeptidase digestions to produce sequenceladders. This was particularly useful where one peptide may harbor twoor more phosphorylation sites.

Panning on Phosphorylated GST-Fusion Proteins.

GST fusion proteins were phosphorylated in vitro as described (Schmitzet al., 1996; Dente et al., 1997; Gram et al., 1997). Briefly, 10 μg/mlwas incubated for 3 h at room temperature with 5.5 units of Fyn proteinkinase in reaction buffer (50 mM Tris, 5 mM MgCl₂, 500 μM Na₃VO₄, 500 μMATP in a total volume of 50 μl). The reaction was stopped by adding 40%of TCA. After the kinase substrate protein was precipitated, it wasresuspended in PBS and coated on microtiter wells at 10 μg/well. Analiquot of CX₇C library (2.5×10¹¹ transducing units) was incubated onthe GST fusion proteins. Phage were sequenced from randomly selectedclones.

Mass Spectrometry Studies

Mass spectrometric peptide mass mapping was used to identify novelligands for β3 and/or β5 cytoplasmic domains. Polyclonal and monoclonalantibodies raised against the cytoplasmic domain-binding peptides wereused to purify target proteins (cytoskeletal or signaling molecules).These proteins were resolved by SDS-PAGE, cut out from the SDS gels, anddigested in-gel with trypsin. After extraction of the peptides,MALDI-TOF mass spectrometry analysis was performed to produce a list ofpeptide masses. This list of peptide masses, in combination withprotease specificity, produces a relatively specific “signature” thatcan be used to search sequence databases. If the protein sequence ispresent in a database, the protein can be identified with highconfidence by this method. The lower detection limit for this approachis currently 1 pmol; at least 10-20-fold better than N-terminal Edmansequencing methods.

Results

Panning of Phage Peptide Libraries on β3 or β5 Cytoplasmic Domains.

β3 and β5 cytoplasmic domain-binding peptides were isolated by screeningmultiple phage libraries with recombinant GST fusion proteins thatcontained either GST-β3cyto or GST-β5cyto coated onto microtiter wells.Immobilized GST was used as a negative control for enrichment during thepanning of each cytoplasmic domain. Phage were sequenced from randomlyselected clones after three rounds of panning as disclosed elsewhere(Koivunen et al., Biotechnology 13:265-270, 1995; Pasqualini et al.,1995). Distinct sequences were isolated that interacted specificallywith the β3 or with the β5 cytoplasmic domains (Table 4). Randomlyselected clones from panning rounds II and III were sequenced. Aminoacid sequences of the phagemid encoded peptides were deduced fromnucleotide sequences. The most frequent motifs found after panning withthe indicated libraries are shown in Table 4. The ratios were calculatedby dividing the number of colonies recovered from β3-GST-coated wellsand those recovered from GST or BSA.

TABLE 4 Sequences displayed by phage binding to β3 or β5 integrincytoplasmic domain β3/GST β3/BSA Peptide motif SEQ ID NO Ratio RatioCX₉ Library CEQRQTQEGC SEQ ID NO:27 4.3 14 CARLEVLLPC SEQ ID NO:28 2.818.7 X₄YX₄ Library YDWWYPWSW SEQ ID NO:29 5.6 163 GLDTYRGSP SEQ ID NO:304.1 48 SDNRYIGSW SEQ ID NO:31 3.3 32 YEWWYWSWA SEQ ID NO:32 2.2 28.1KVSWYLDNG SEQ ID NO:33 2.1 20 SDWYYPWSW SEQ ID NO:34 2.1 157 AGWLYMSWKSEQ ID NO:35 1.8 2.4 Pool Cyclic Libraries CFQNRC SEQ ID NO:36 3.1 16CNLSSEQC SEQ ID NO:37 2.7 62 CLRQSYSYNC SEQ ID NO:38 2.4 3.2 β5/GSTβ5/BSA Peptide motif SEQ ID NO Ratio Ratio Pool Cyclic LibrariesCYIWPDSGLC SEQ ID NO:39 5.2 193 CEPYWDGWFC SEQ ID NO:40 3.1 400CKEDGWLMTC SEQ ID NO:41 2.3 836 CKLWQEDGY SEQ ID NO:42 1.8 665CWDQNYLDDC SEQ ID NO:43 1.5 100 X₄YX₄ Library DEEGYYMMR SEQ ID NO:4411.5 29 KQFSYRYLL SEQ ID NO:45 4.5 8 VVISYSMPD SEQ ID NO:46 3.8 28SDWYYPWSW SEQ ID NO:34 2.4 304

The specificity of the interaction with β3 or β5 cytoplasmic domains wasdetermined by calculating the ratios of phage bound to the cytoplasmicdomain containing—fusion proteins (β3 or β5) versus GST alone (negativecontrol). FIG. 18 shows the results from binding assays performed withthe GST-β3cyto binding phage. Six phage were tested that displayed themotifs most frequently found during the second and third rounds ofpanning. Each panel shows the results from binding assays for the phagedisplaying different peptides that bind to the β3 cytoplasmic domain, asindicated. Insertless phage or unselected libraries were used asnegative controls and did not show binding above background. Two platingdilutions were shown for each assay.

A similar strategy was used to determine the specificity of the phageisolated in the screenings involving the β5 cytoplasmic domain fusionprotein. The binding assays were performed with individually amplifiedphage, shown in FIG. 19. Five phage were tested that displayed themotifs found most frequently during the second and third rounds ofpanning. Each panel shows the binding assays for the phage displayingpeptides that bind to the β5 cytoplasmic domain. Insertless phage orunselected libraries were used as negative controls and did not showbinding above background in these assays.

To determine whether the binding of the selected motifs was specific foreach cytoplasmic domain, binding assays were performed comparing theinteraction of individual phage motifs with β1, β3, or β5 cytoplasmicdomain fusion proteins. ELISA with anti-GST antibodies showed that thethree proteins can be coated onto plastic at equivalent efficiency, andthus the differences in binding do not reflect differences in coatingconcentrations (not shown). Both the β3- and β5-selected phageselectively interacted with the proteins on which they were originallyselected, with average binding selectivities observed of β3/β1=3.9,β3/β5=3.7, β5/β1=4.8, and β5/β3=6.9 (not shown). The average selectivityfor integrin cytoplasmic domains versus BSA was about one to two ordersof magnitude (not shown). None of the phage tested seemed to bindstrongly to the 131 cytoplasmic domain (not shown).

Characterization of Synthetic Peptides Corresponding to the SequencesDisplayed by the Integrin-Cytoplasmic Domain-Binding Phage.

Specific phage were selected for further studies on the basis of theirbinding properties. Synthetic peptides corresponding to the sequencedisplayed by each phage were used to perform binding inhibition studies.This assay determined whether phage binding was entirely mediated by thetargeting peptide displayed by the phage or whether it also included anon-specific component. As expected, the synthetic peptides inhibitedthe binding of the corresponding phage in a dose-dependent manner (FIG.20 and FIG. 21). A control peptide containing unrelated amino acids hadno effect on phage binding when tested at identical concentrations.

Phosphorylation Events Modulate the Interaction of the Selected Peptideswith Cytoplasmic Domains

Events involving phosphorylation are important in regulating signaltransduction. The phage display system was used to evaluate the effectof tyrosine phosphorylation at two levels. First, recombinant fusionproteins containing β3 or β5 cytoplasmic domains were used for panningof phage libraries displaying tyrosine-containing peptides. Second, thecytoplasmic domains themselves were phosphorylated before phageselection was performed. Experiments were performed to investigate thecapacity of specific tyrosine kinases to modulate the interaction of theselected peptides with the cytoplasmic domains. The results obtained inthe panning of phage libraries displaying tyrosine-containing peptidesare shown in Table 5.

Randomly selected clones from rounds III and IV were sequenced from aX₄YX₄ phosphorylated library with Fyn. Amino acid sequences of thephagemid encoded peptides were deduced from nucleotide sequences. Table5 shows the motifs found most frequently after the indicated librarieswere panned with β3 or β5. The ratio of binding to β3 or β5 wascalculated by dividing the number of β3 or β5 colonies by GST or BSAcolonies found after panning. The ratio of binding to β3 or β5 withphosphorylated phage by Fyn versus unphosphorylated phage was calculatedby dividing the number of colonies found after the panning.

TABLE 5 Sequences displayed by phosphorylated phage binding to integrincytoplasmic domains. Phos/ β3 or β3 or Peptide Motif Unphos β5/GSTβ5/BSA β3 cytoplasmic GGGSYRHVE SEQ ID NO:49 13.2 1.5 5.3 RAILYRLAN SEQID NO:50 2.8 1.3 20 MLLGYRFEK SEQ ID NO:51 2.5 3.5 2.7 β5 cytoplasmicTMLRYTVRL SEQ ID NO:52 14.3 3.4 2.2 TMLRYFMFP SEQ ID NO:53 4.2 2.3 3.8TLRKYFHSS SEQ ID NO:54 3.8 3.8 15.2

The effect of phosphorylation on the affinity and specificity of thecytoplasmic domain-binding was examined. Phage displaying the β3 and β5cytoplasmic domain-binding peptides were phosphorylated in vitro aspreviously described (Schmitz et al., 1996; Dente et al., 1997; Gram etal., 1997), using Fyn kinase. Specific phosphorylation of thetyrosine-containing peptide on the surface of the phage was confirmed byusing ³²P-gamma DATP in the kinase reaction and by separating the phagepIII protein by SDS-PAGE.

Phage phosphorylated in vitro showed increased binding affinity andspecificity to the β3 integrin cytoplasmic domain (FIG. 22). TheTLRKYFHSS (SEQ ID NO:54) phage was also tested in assays that includedother GST-cytoplasmic domain fusion proteins to determine specificity(FIG. 23).

Sequence Similarity of Integrin Binding Peptides with Known Cytoskeletaland Signaling Proteins.

The peptides displayed by integrin cytoplasmic domain-binding phage weresimilar to certain regions found within cytoskeletal proteins andproteins involved in signal transduction (Table 6). The similarity ofsome of the isolated peptides to a region of mitogen-activated proteinkinase 5 (MAPK5, amino acids 227-234) was particularly interesting. Aconnection involving the MAPK cascade, cell adhesion, migration andproliferation has been proposed (Lin et al., 1997)

TABLE 6 Sequence similarity of integrin binding peptides with knowncytoskeletal and signaling proteins. Region Homology Isolated MotifCandidate Proteins (AA #) % β3 cytoplasmic GLDTYRGSP Ras-related protein124-133 75 (SEQ ID NO:30) Ser/Thr kinase (K-11) 18-25 75 SDNRYIGSW PDGFreceptor 985-992 85 (SEQ ID NO:31) Phosphatidylinositol 233-241 85 4phosphatase 5 Receptor protein kinase 185-191 85 Protein kinase clk271-79 63 CEQRQTQEGC MAPK5 227-234 75 (SEQ ID NO:27) Phosphatidylinositol494-503 78 3-kinase CLRQSYSYNC Cyclin-dependent kinase 230-239 75 (SEQID NO:38) 5 (cdk5) β5 cytoplasmic VVISYSMPD Ser/Thr kinase 479-485 83(SEQ ID NO:46) IFN (β chain) 27-35 70 Actin 240-248 67 DEEGYYMMR Focaladhesion kinase 43-51 75 (SEQ ID NO:44) Tubulin 60-66 100 PutativeSer/Thr kinase 292-299 86

Membrane-Permeable Peptides

Penetratin is a peptide that can translocate hydrophilic compoundsacross the plasma membrane. Fusion to the penetrating moiety allowsoligopeptides to be targeted directly to the cytoplasm, nucleus, or bothwithout apparent degradation (Derossi et al., 1994). Thismembrane-permeable peptide consists of 16 residues (RQIKIWFQNRRMKWKK,SEQ ID NO:55) corresponding to amino acids 43-58 of the homeodomain ofAntennapedia, a Drosophila transcription factor (Joliet et al., 1991a,1991b; Le Roux et al., 1993). Internalization mediated by penetratinoccurs at both 37° C. and 4° C., and the internalized peptide can beretrieved intact from cells.

Peptides were designed containing penetratin sequences fused to thesequences of motifs found to bind β3 or β5 cytoplasmic domains. Thepeptides were synthesized on a 431 Applied Biosystems peptidesynthesizer using p-hydroxymethylphenoxy methyl polystyrene (HMP) resinand standard Fmoc chemistry. Peptide internalization and visualizationwas performed as described (Derossi et al., 1994; Hall et al., 1996;Theodore et al., 1995).

Briefly, 10-50 μg/ml of the biotinylated peptide was added to cells inculture. Peptides were incubated with plated cells. After 2-4 hours, thecultures were washed three times with tissue culture media, fixed andpermeabilized using ethanoi:acetic acid (9:1) for 5 min at −20° C.Nonspecific protein binding sites were blocked by incubating thecultures for 30 min with Tris-buffered saline (TBS) containing 10% fetalcalf serum (FCS) and 0.02% Tween. The cultures were incubated in thesame buffer containing FITC-conjugated Streptavidin (1:200 dilution) andwashed with TBS before being mounted for viewing by confocal microscopy.The penetratin-linked peptides were internalized quite efficiently (datanot shown).

Functional data showed that the cytoplasmic domain-binding peptidesselected on β3 or β5 can interfere with integrin-mediated signaling andsubsequent cellular responses (i.e., endothelial cell adhesion,spreading, proliferation, migration). A commercial panel of“internalizable” versions of the synthetic motifs found by phagescreenings (SDNRYIGSW (SEQ ID NO:31); CEQRQTQEGC (SEQ ID NO:27); B3binding peptides and VVISYSMPD (SEQ ID NO:46); a β5-binding peptide)were obtained. These complex chimeric peptides consist of the mostselective of the β3 or β5-cytoplasmic domain-binding peptides coupled topenetratin, plus a biotin moiety to allow the peptides to be trackedonce they were internalized into intact cells. These membrane-permeableforms of the peptides are internalized, may affect β3 and β5 post-ligandbinding cellular events and can induce massive apoptosis (data notshown).

Endothelial Cell Proliferation, Chemotaxis and Apoptosis

The effect of β3 and β5 integrin cytoplasmic domain-binding motifs onendothelial cell proliferation was evaluated after stimulation withfactors that activate endothelial cells (FIG. 24). Cell proliferationwas measured according to Pasqualini and Hemler (1994). Briefly, 4×10⁴HUVECs were incubated in 24-well plates and starved for 24 h, afterwhich the medium was removed and replaced in the presence of VEGF and 15μM of each peptide. After another 18 h of incubation, 50 μl of mediumcontaining [³H]thymidine (1 μCi/ml) was added to the wells. After 6additional hours of incubation at 37° C., the medium was removed and thecells were fixed in 10% TCA for 30 min at 4° C., washed with ethanol andsolubilized in 0.5 N NaOH. Radioactivity was counted by liquidscintillation by using a LS 6000SC Beckman scintillation counter. Eachexperiment was performed three times with triplicates, and the resultswere expressed as the mean±SD.

The effect of β3 and β5 integrin cytoplasmic domain-binding motifs inendothelial cell migration was evaluated after stimulation with factorsthat activate endothelial cells. The peptides tested affected cellfunction in a dose-dependent and specific way. Their properties seem tobe intrinsic to the β3 or to the β5 cytoplasmic domain (FIG. 25).

Chemotaxis Assay.

Cell migration was assayed in a 48-well microchemotaxis chamber.Polyvinylpyrrolidone-free polycarbonate filters with 8-μm pores werecoated with 1% gelatin for 10 min at room temperature and equilibratedin M199 medium supplemented with 2% FCS. Peptides were placed in thelower compartment of a Boyden chamber in M199 supplemented with 2% FCS,20 ng/ml VEGF-A (R&D System), and 1 U/ml heparin. Overnight-starvedsubconfluent cultures were quickly trypsinized, and resuspended in M199containing 2% FCS at a final concentration of 2×10⁶ cells/ml. After thefilter was placed between lower and upper chambers, 50 μl of the cellsuspension was seeded in the upper compartment. Cells were allowed tomigrate for 5 h at 37° C. in a humidified atmosphere with 5% CO₂. Thefilter was then removed, and cells on the upper side were scraped with arubber policeman. Migrated cells were fixed in methanol and stained withGiemsa solution. Five random high-power fields (magnitude 40×) werecounted in each well. The results showed that both β3-integrincytoplasmic domain binding peptides increased cell migration butpenetratin did not affect the cells (data not shown).

Apoptosis Assay (Propidium Iodide (PI) Staining Subdiploid Population).

Approximately 1×10⁶ cells were harvested in complete medium, and 15 μMof peptide was added for 4, 8, or 12 hours. The cells were then washedin PBS and resuspended in 0.5 ml propidium iodide solution (50 μg/ml PI,0.1% Triton X-100, 0.1% sodium citrate). After a 24-h incubation at 4°C., the cells were counted with an XL Coulter (Coulter Corporation) witha 488 nm laser; 12,000 cells were counted for each histogram, and cellcycle distributions were analyzed with the Multicycle program.

Treatment of cells with VISY-penetratin chimera resulted in induction ofapoptosis (FIG. 26, panel d). Pro-apoptotic effects were not observedwhen the cells were exposed to other growth factors (not shown).Penetratin alone and the other penetratin chimeras also could not inducesimilar effects (FIG. 26, panel c). This finding shows that novelapproaches for inhibiting angiogenesis can be developed based on the useof integrin targeting peptides.

Immunization with Cytoplasmic Domain Binding Peptides andCharacterization of the Resulting Antibodies

Polyclonal antibodies that recognize αvββ3 and αvβ5-binding peptideswere generated using KLH conjugates made with the synthetic peptides,according to standard techniques. Antibodies against two differentsynthetic peptides have been produced (FIG. 27). The sera not onlyrecognize the immobilized peptides, but also recognize specific proteinsin total cell extracts, as shown by western blot analysis (FIG. 28).

Rabbits were immunized with SDNRYIGSW (SEQ ID NO:31) or GLDTYRGSP (SEQID NO:30)-KLH conjugates. Each rabbit was injected with 200 μg ofpeptide conjugated with KLH in Complete Freund's Adjuvant. Between 20and 60 days later, the rabbits were injected with 100 μg IncompleteFreund's Adjuvant. After the third immunization, sera was collected.Pre-immune serum obtained before the first immunization was used as anadditional control in the experiments.

The polyclonal antibodies were tested by ELISA, Western blot andimmunoprecipitation. In the ELISA assays, microtiter well plates werecoated with 10 μg/ml of peptides. The plates were dried at 37° C.,blocked with PBS+3% BSA, and incubated with different serum dilutions inPBS+1% BSA. After washing and incubation with the secondary antibody, analkaline phosphate substrate was added and antibody binding detectedcolorimetrically at 405 nm. The reactivity observed both in the mouseand rabbit polyclonal sera was highly specific. In all cases, antibodybinding could be abrogated by preincubation with the correspondingpeptide that was used for immunization, but not by a control peptide(FIG. 27 and FIG. 28). Antibodies raised against two of the β3cytoplasmic domain binding peptides recognize specific bands on totalcell extracts and in immunoprecipitation experiments using 35S-labeledextracts. Similar results were obtained with polyclonal sera andpurified IgGs (not shown).

The present example shows that targeting peptides against specificdomains of cell receptors can be identified by phage display. Suchpeptides may be used to identify the endogenous ligands for cellreceptors, such as endostatin. In addition, the peptides themselves mayhave therapeutic effects, or may serve as the basis for identificationof more effective therapeutic agents. The endostatin targeting peptidesidentified herein, when introduced into cells, showed effects on cellproliferation, chemotaxis and apoptosis. The skilled artisan willrealize that the present invention is not limited to the disclosedpeptides or therapeutic effects. Other cell receptors and ligands, aswell as inhibitors or activators thereof, may be identified by thedisclosed methods.

Example 7 Induction of Apoptosis with Integrin Binding Peptides(Endothanos)

Example 6 showed that the VISY peptide (VVISYSMPD, SEQ ID NO:46),imported into cells by attachment to penetratin, could induce apoptosisin HUVEC cells. Antibodies raised against the VISY peptide were used toidentify the endogenous cell analog of the peptide, identified herein asAnnexin V. The results indicate that Annexin V is an endogenous ligandfor the integrins that is involved in a novel pathway for apoptosis.

Methods

Protein Purification

Polyclonal antibodies against the VISY peptide (VVISYSMPD, SEQ ID NO:46)were prepared using the methods described in Example 6. MDA-MB-435breast carcinoma cells were used for purification of the endogenous VISYpeptide analog. Cells were washed three times with ice cold PBS andlysed with chilled water for 20 min. Cell extracts were centrifuged for30 min at 100,000×g to separate the cytoplasmic fraction from themembrane fraction. The cytoplasmic fraction was subjected to columnchromatography on a gel filtration column (10-50 kDa) and an anionexchange column (mono Q). The anion exchange column was eluted with asalt gradient from 50 mM to 1 M NaCl. One ml fractions were collected,run on SDS-PAGE and tested by Western blotting for the presence ofendogenous proteins reactive with the anti-VISY antibody. The fractionof interest, containing a 36 kDa antibody reactive band, eluted at about300 mM NaCl.

The 36 kDa always appeared in fractions that showed positive reactivitywith the anti-VISY antibody. The fractions were analyzed by SDS-PAGE and2-D gel electrophoresis, followed by Western blotting. A substantialenrichment of the 36 kDa protein was seen after column chromatography(not shown). The 36 kDa peptide was cut from the SDS-PAGE gel andanalyzed by mass spectroscopy to obtain its sequence. All five peptidesequences that were obtained by mass spectroscopy showed 100% homologyto the reported sequence of Annexin V (GenBank Accession No.GI_(—)468888). In addition to its presence in 435 cells, the 36 kDa bandwas also seen in Kaposi sarcoma, SKOV and HUVEC cells (not shown).

Commercial antibodies against Annexin V were obtained (Santa CruzBiologics, Santa Cruz, Calif.). Comparative Western blots were performedusing the anti-VISY antibody and the anti-Annexin V antibody. Bothantibodies showed reactivity with the 36 kDa protein (not shown). Theseresults indicate that the endogenous protein analog of the VISY peptideis Annexin V.

Protein-Protein Interaction with Annexin V and β5 Cytoplasmic Domain.

Competitive binding assays were performed to examine the binding ofAnnexin V to β5 integrin and the effect of the VISY peptide. Plates werecoated with GST fusion proteins of the cytoplasmic domains of variousintegrins and Annexin V was added to the plates. Binding of Annexin Vwas determined using anti-Annexin V antibodies. As shown in FIG. 29A,Annexin V did not bind to either the GST-β1 or GST-β3 integrins; AnnexinV bound strongly to the GST-β5 integrin, but binding was dependent onthe buffer used (FIG. 29A). Low binding was observed in Tris-bufferedsaline (TBS), while high binding was observed in “cytoplasmic buffer”(100 mM KCl, 3 mM NaCl, 3.5 mM MgCl₂, 10 mM PIPES, 3 mM DTT) with orwithout added calcium (2 μM) (FIG. 29A). Calcium was used becauseAnnexin V activity has been reported to be modulated by calcium. Bindingof Annexin V to GST-β5 was blocked by addition of the VISY peptide (FIG.29A). FIG. 29B shows the relative levels of binding of anti-Annexin Vantibody to purified Annexin V and to VISY peptide.

A reciprocal study was performed, using Annexin V to coat plates andadding GST fusion proteins of integrin cytoplasmic domains. Binding wasassessed using anti-GST fusion protein antibodies. As expected, onlyGST-β5 showed substantial binding to Annexin V, while GST-β1 and GST-β3showed low levels of Annexin V binding (not shown). In some studies,calcium ion appeared to interfere with the binding interaction betweenGST-β5 and Annexin V, with decreased binding observed in the presence ofcalcium (not shown). A greater degree of inhibition of Annexin V bindingto GST-β5 by the VISY peptide was observed in the presence of calcium(67% inhibition) than in the absence of calcium (45%) (FIG. 29A).

Penetratin Peptide Chimera Binding to the β5 Cytoplasmic Domain InducesProgrammed Cell Death.

The induction of apoptosis by VISY peptide was shown in Example 6 wasconfirmed. 10⁶ HUVEC were treated with 15 μM of VISY antennapedia(penetratin) chimera or 15 μM of antennapedia peptide (pentratin) alonefor 24 hours and chromatin fragmentation was analyzed by electrophoresisin an agarose gel. FIG. 30 shows the induction of apoptosis by VISY-Ant(penetratin), as indicated by chromatin fragmentation. Neither VISY norpenetratin alone induced apoptosis. Induction of apoptosis was inhibitedup to 70% when a caspase inhibitor (ZVAD, caspase inhibitor I,Calbiochem #627610, San Diego, Calif.) was added to the media at thesame time as the VISY chimeric peptide.

A distinction between the mechanism of cell death induced by VISYpeptide and other pro-apoptosis agents is that other apoptoticmechanisms evaluated in cell culture typically involve detachment of thecells from the substrate, followed by cell death. In contrast, in VISYinduced cell death, the cells do not detach from the substrate beforedying. Thus, endothanos (death from inside) appears to differ fromanoikis (homelessness).

The present results show that VISY peptides activate an integrindependent apoptosis pathway. The present example shows that theendogenous analog for VISY peptide is Annexin V. These resultsdemonstrate the existence of a novel apoptotic pathway, mediated throughan interaction between Annexin V and β5 integrin and dependent oncaspase activity. This novel apoptotic mechanism is termed endothanos.The skilled artisan will realize that the existence of a novel mechanismfor inducing or inhibiting apoptosis is of use for a variety ofapplications, such as cancer therapy.

Example 8 Identification of Receptor/Ligand Pairs: Aminopeptidase ARegulates Endothelial Cell Function and Angiogenesis

Endothelial cells in tumor vessels express specific angiogenic markers.Aminopeptidase A. (APA, EC 3.4.11.7) is upregulated in microvesselsundergoing angiogenesis. APA is a homodimeric, membrane-bound zincmetallopeptidase that hydrolyzes N-terminal glutamyl or aspartylresidues from oligopeptides (Nanus et al., 1993). In vivo, APA convertsangiotensin II to angiotensin III. The renin-angiotensin system plays animportant role in regulating several endocrine, cardiovascular, andbehavioral functions (Ardaillou, 1997; Stroth and Unger, 1999). Recentstudies also suggest a role for angiotensins in angiogenesis (Andrade etal., 1996), but the function of APA in the angiogenic process has notbeen investigated so far.

In the present example, targeting peptides capable of binding APA wereidentified by screening phage libraries on APA-expressing cells.APA-binding peptides containing the motif CPRECESIC (SEQ ID NO:56)specifically inhibited APA enzyme activity. Soluble CPRECESIC (SEQ IDNO:56) peptide inhibited migration, proliferation, and morphogenesis ofendothelial cells in vitro and interfered with in vivo angiogenesis in achick embryo chorioallantoic membrane (CAM) assay. Furthermore, APA nullmice had a decreased amount of retinal neovascularization compared towild type (wt) mice in hypoxia-induced retinopathy in premature mice.These results may lead to a better understanding of the role of APA inangiogenesis and to development of new anti-tumor therapeuticstrategies.

Materials and Methods

Cell Cultures

The renal carcinoma cell line SK-RC-49 was transfected with anexpression vector encoding full-length APA cDNA (Geng et al., 1998).Cells were maintained in MEM (Irvine Scientific, Santa Ana, Calif.),supplemented with 2 mM glutamine, 1% nonessential amino acids, 1%vitamins (Gibco BRL), 100 U/ml streptomycin, 100 U/ml penicillin (IrvineScientific), 10 mM sodium pyruvate (Sigma-Aldrich), and 10% fetal calfserum (FCS) (Tissue Culture Biological, Tulare, Calif.). Stablytransfected cells were maintained in G418-containing medium. HUVECs wereisolated by collagenase treatment and used between passages 1 to 4.Cells were grown on gelatin-coated plastic in M199 medium (Sigma)supplemented with 20% FCS, penicillin (100 U/ml), streptomycin (50μg/ml), heparin (50 μg/ml), and bovine brain extract (100 μg/ml). Allmedia supplements were obtained commercially (Life Technologies, Inc.,Milan, Italy).

Antibodies and Peptides

The anti-APA nAb RC38 (Schlingemann et al., 1996) was used toimmunocapture APA from transfected cell lysates. CPRECESIC (SEQ. IDNO:56) and GACVRLSACGA (SEQ ID NO:57) cyclic peptides were chemicallysynthesized, spontaneously cyclized in non-reducing conditions, andpurified by mass spectrometry (AnaSpec San Jose, Calif.). The massspectrometer analysis of the CPRECESIC (SEQ ID NO:56) peptide revealedsix different peaks, possibly reflecting different positions ofdisulfide bounds and the formation of dimers. Due to the similarbiochemical behavior of the different fractions on APA enzyme activity,a mix of the six peaks was used in all procedures described below.

APA Immunocapture

Cells were scraped from semi-confluent plates in cold PBS containing 100mM N-octyl-β-glucopyranoside (Calbiochem), lysed on ice for 2 h, andcentrifuged at 13,000×g for 15 min. Microtiter round-bottom wells(Falcon) were coated with 2 μg of RC38 for 4 h at room temperature andblocked with PBS/3% BSA (InterGen, Purchase, N.Y.) for 1 h at roomtemperature, after which 150 μl of cell lysate (1 mg/ml) was incubatedon the mAb-coated wells overnight at 4° C., washed five times withPBS/0.1% Tween-20 (Sigma), and washed twice with PBS.

APA Enzyme Assay

Cells and immunocaptured proteins were tested for specific enzymeactivity according to Liln et al. (1998). Briefly, adherent cells orRC38-immunocaptured cell extracts were incubated for 2 h at 37° C. withPBS containing 3 mM α-L-glutamyl-p-nitroamide (Fluka) and 1 mM CaCl₂.Enzyme activity was determined by reading the optical absorbance (O.D.)at 405 nm in a microplate reader (Molecular Devices, Sunnyvale, Calif.).

Cell Panning

A CX₃CX₃CX₃C(C, cysteine; X, any amino acid) library was prepared(Rajotte et al., 1998). Amplification and purification of phageparticles and DNA sequencing of phage-displayed inserts were performedas described. Cells were detached by incubation with 2.5 mM EDTA in PBS,washed once in binding medium (DMEM high glucose supplemented with 20 mMHEPES and 2% FCS), and resuspended in the same medium at a concentrationof 2×10⁶ cells/ml. 10¹⁰ TU of phage were added to 500 μl of the cellsuspension, and the mixture was incubated overnight (first round) or for2 h (successive rounds) at 4° C. with gentle rotation. Cells were washedfive times in binding medium at room temperature and resuspended in 100μl of the same medium. Phage were rescued by adding 1 ml ofexponentially growing K91Kan Escherichia coli bacteria and incubatingthe mixture for 1 h at room temperature. Bacteria were diluted in 10 mlof LB medium supplemented with 0.2 μg/ml tetracycline and incubated foranother 20 min at room temperature. Serial dilutions were plated on LBplates containing 40 μg/ml tetracycline, and plates were incubated at37° C. overnight before colonies were counted.

Phage Binding Specificity Assay

The cell binding assay was performed with an input of 10⁹ TU asdescribed for the cell panning. The specificity was confirmed by addingCPRECESIC (SEQ ID NO:56) peptide to the binding medium in increasingconcentrations. For phage binding on immunocaptured APA, wells wereblocked for 1 h at room temperature with PBS/3% BSA and incubated with10⁹ TU for 1 h at room temperature in 50 μl PBS/3% BSA. After eightwashes in PBS/1% BSA/0.01% Tween-20 and two washes in PBS, phage wererescued by adding 200 μl of exponentially growing K91Kan E. coli. Eachexperiment was repeated at least three times.

In Vivo Tumor Homing of APA-Binding Phage

MDA-MB-435-derived tumor xenografts were established in female nude mice2 months old (Jackson Labs; Bar Harbot, Me.). Mice were anesthetizedwith Avertin and injected intravenously through the tail vein with 10⁹TU of the phage in a 200 μl volume of DMEM. The phage were allowed tocirculate for 5 min, and the animals were perfused through the heartwith 5 ml of DMEM. The tumor and brain were dissected from each mouse,weighed, and equal amounts of tissue were homogenized. The tissuehomogenates were washed three times with ice-cold DMEM containing aproteinase inhibitor cocktail and 0.1% BSA. Bound phage were rescued andcounted as described for cell panning. Fd-tet phage was injected at thesame input as a control. The experiment was repeated twice. In parallel,part of the same tissue samples were fixed in Bouin solution, andimbedded in paraffin for preparation of tissue sections. An antibody toM-13 phage (Amersham-Pharmacia) was used for the staining.

Cell Growth Assay

HUVECs were seeded in 48-well plates (10⁴ cells/well) and allowed toattach for 24 h in complete M199 medium. The cells were then starved inM199 medium containing 2% FCS for 24 h. CPRECESIC (SEQ ID NO:56) orcontrol GACVRLSACGA (SEQ ID NO:57) peptide (1 mM) was added to the wellsin medium containing 2% FCS and 10 ng/ml VEGF-A (R&D System, Abingdom,UK). After incubation for the indicated times, cells were fixed in 2.5%glutaraldehyde, stained with 0.1% crystal violet in 20% methanol, andsolubilized in 10% acetic acid. All treatments were done in triplicate.Cell growth was evaluated by measuring the O.D. at 590 nm in amicroplate reader (Bio-Rad Laboratories, Hercules, Calif.). Acalibration curve was established and a linear correlation between O.D.and cell counts was observed between 10³ and 10⁵ cells.

Chemotaxis Assay

A cell migration assay was performed in a 48-well microchemotaxischamber (NeuroProbe, Gaithersburg, Md.) according to Bussolini et al.(1995). Polyvinylpyrrolidone-free polycarbonate filters (Nucleopore,Cambridge, Mass.) with 8-μm pores were coated with 1% gelatin for 10 minat room temperature and equilibrated in M199 medium supplemented with 2%FCS. CPRECESIC (SEQ ID NO:56) or control GACVRLSACGA (SEQ ID NO:57)peptide (1 mM) was placed in the lower compartment of a Boyden chamberin M199 medium supplemented with 2% FCS and 10 ng/ml VEGF-A (R&DSystem). Subconfluent cultures that had been starved overnight wereharvested in PBS containing 2.5 mM EDTA, washed once in PBS, andresuspended in M199 medium containing 2% FCS at a final concentration of2×10⁶ cells/ml. After the filter was placed between the lower and upperchambers, 50 μl of the cell suspension was seeded in the uppercompartment, and cells were allowed to migrate for 5 h at 37° C. in ahumidified atmosphere with 5% CO₂. The filter was then removed, andcells on the upper side were scraped with a rubber policeman. Migratedcells were fixed in methanol and stained with Giemsa solution(Diff-Quick, Baxter Diagnostics, Rome, Italy). Five random high-powerfields (magnitude 100×) were counted in each well. Each assay was run intriplicate.

Three-Dimensional Cell Culture

Matrigel (Collaborative Research, Bedford, Mass.) was added at 100 μlper well to 48-well tissue culture plates and allowed to solidify for 10min at 37° C. HUVECs were starved for 24 h in M199 medium supplementedwith 2% FCS before being harvested in PBS containing 2.5 mM EDTA. 10⁴cells were gently added to each of the triplicate wells and allowed toadhere to the gel coating for 30 min at 37° C. Then, medium was replacedwith indicated concentrations of CPRECESIC (SEQ ID NO:56) or GACVRLSACGA(SEQ ID NO:57) peptides in complete medium. The plates were photographedafter 24 h with an inverted microscope (Canon). The assay was repeatedthree times.

CAM Assay

In vivo angiogenesis was evaluated by a CAM assay (Ribatti et al.,1994). Fertilized eggs from White Leghorn chickens were maintained inconstant humidity at 37° C. On the third day of incubation, a squarewindow was opened in the eggshell and 2-3 ml of albumen was removed todetach the developing CAM from the shell. The window was sealed with aglass plate of the same size and the eggs were returned to theincubator. At day 8, 1 mm³ sterilized gelatin sponges (Gelfoam, UpjohnCo, Kalamazoo, Milan) were adsorbed with VEGF-A (20 ng, R&D System) andeither CPRECESIC (SEQ ID NO:56) or control GACVRLSACGA (SEQ ID NO:57)peptide (1 mM) in 3 μl PBS and implanted on the top of the growing CAMsunder sterile conditions. CAMs were examined daily until day 12 andphotographed in ovo with a Leica stereomicroscope. Capillaries emergingfrom the sponge were counted. The assay was repeated twice.

Induction of Retinal Neovascularization

APA null mice have been described (Lin et al., 1998). Mice pups on P7(7^(th) day postpartum) with their nursing mothers were exposed to 75%oxygen for 5 days. Mice were brought back to normal oxygen (room air) onP12. For histological analysis mice were killed between P17 and P21 andeyes were enucleated and fixed in 4% paraformaldehyde in PBS overnightat 4° C. Fixed eyes were imbedded in paraffin and 5 μm serial sectionswere cut. Sections were stained with hematoxylin/eosin solution.Neovascular nuclei on the vitreous side of the internallimiting-membrane were counted from 20 h/e-stained sections per eacheye. The average number of neovascular nuclei per section was calculatedand compared between animal groups using Student's t-test.

Results

Cell Panning with Phage Display Select an APA-Binding Motif

To identify a peptide capable of binding to APA, cells were screenedwith a random peptide phage library. First, SK-RC-49 renal carcinomacells, which do not express APA, were transfected with full-length APAcDNA to obtain a model of APA expression in the native conformation. APAexpressed as a result of transfection was functionally active, asevidenced by an APA enzyme assay (not shown), but parental SK-RC-49cells showed neither APA expression nor activity (not shown).

The CX₃CX₃CX₃C phage library (10¹⁰ transducing units [TU]) waspreadsorbed on parental SK-RC-49 cells to decrease nonspecific binding.Resuspended SK-RC-49/APA cells were screened with phage that did notbind to the parent cells. SK-RC-49/APA-bound phage were amplified andused for two consecutive rounds of selection. An increase in phagebinding to SK-RC-49/APA cells relative to phage binding to SK-RC-49parental cells was observed in the second and third rounds (not shown).

Subsequent sequencing of the phage revealed a specific enrichment of apeptide insert, CYNLCIRECESICGADGACWTWCADGCSRSC (SEQ ID NO:58), with atandem repetition of the general library sequence CX₃CX₃CX₃C. Thissequence represented 50% of 18 randomly selected phage inserts fromround 2 and 100% of phage inserts from round 3. Four peptide insertsderived from round 2 shared sequence similarity with the tandem phage(Table 7, in bold font). Several other apparently conserved motifs wereobserved among round 2 peptides (Table 7, underlined or italicized). Oneof these overlapped in part with the tandem repeated sequence. A searchfor sequence homology of the selected peptides against human databasesdid not yield a significant match.

TABLE 7 APA-binding peptide sequences. Peptide sequences Round 2 (%)/3(%) CYNLCIRECESICGADGACWTWCADGCSRSC (SEQ ID NO:58) 50/100 CLGQCASICVNDC(SEQ ID NO:59)  5/− CPKVCPRECESNC (SEQ ID NO:60)  5/− CGTGCAV EC EVVC(SEQ ID NO:61)  5/− CAVACWADCQLGC (SEQ ID NO:62)  5/− CSGLCTVQCLEGC (SEQID NO:63)  5/− CSMMCLEGCDDWC (SEQ ID NO:64)  5/− OTHER 20/−

Selected Phage Inserts are Specific APA Ligands.

Phage displaying the peptide inserts CYNLCIRECESICGADGACWTWCADGCSRSC(SEQ ID NO:58), CPKVCPRECESNC (SEQ ID NO:60) or CLGQCASICVNDC (SEQ IDNO:59) were individually tested for APA binding. All three phagespecifically bound to the surface of SK-RC-49/APA cells (not shown),with a similar pattern of 6-fold enrichment relative to SK-RC-49parental cells. Control, insertless phage showed no binding preference(not shown). CGTGCAVECEVVC (SEQ ID NO:61) and the other phage selectedin round 2 showed no selective binding to SK-RC-49/APA cells (data notshown). A soluble peptide, CPRECESIC (SEQ ID NO:56) containing aconsensus sequence reproducing the APA-binding phage inserts wassynthesized.

Binding assays were performed with CPKVCPRECESNC (SEQ ID NO:60) phage inthe presence of the CPRECESIC (SEQ ID NO:56) peptide. Soluble CPRECESIC(SEQ ID NO:56) peptide competed with CPKVCPRECESNC (SEQ ID NO:60) phagefor binding to SK-RC-49/APA cells, but had no effect on nonspecificbinding to SK-RC-49 parental cells (not shown). The unrelated cyclicpeptide GACVRLSACGA (SEQ ID NO:57) had no competitive activity (notshown). Binding of CYNLCIRECESICGADGACWTWCADGCSRSC (SEQ ID NO:58) phagewas also displaced by CPRECESIC (SEQ ID NO:56) peptide, but the bindingof CLGQCASICVNDC (SEQ ID NO:59) phage was not affected (data not shown).

To further confirm the substrate specificity of the selected peptideinserts, APA was partially purified from APA-transfected cell extractsby immunocapture with mAb RC38. The APA protein immobilized onRC38-coated microwells was functional, as confirmed by enzyme assay (notshown). The CYNLCIRECESICGADGACWTWCADGCSRSC (SEQ ID NO:58),CPKVCPRECESNC (SEQ ID NO:60), and CLGQCASICVNDC (SEQ ID NO:59) phageselectively bound immunocaptured APA, with a 10- to 12-fold enrichmentcompared to phage binding to RC38-immunocaptured cell lysates fromSK-RC-49 parental cells (not shown).

APA-Binding Phage Target Tumors In Vivo.

The ability of the identified peptide to home to tumors was evaluated,using nude mice implanted with human breast tumor xenografts as a modelsystem. Phage were injected into the tail vein of tumor-bearing mice,and targeting was evaluated by phage recovery from tissue homogenates.CPKVCPRECESNC (SEQ ID NO:60) phage was enriched 4-fold in tumorxenografts compared to brain tissue, which was used as a control (FIG.31). Insertless phage did not target the tumors (FIG. 31). NeitherCYNLCIRECESICGADGACWTWCADGCSRSC (SEQ ID NO:58) nor CLGQCASICVNDC (SEQ IDNO:59) phage showed any tumor-homing preference (data not shown).

The homing of CPKVCPRECESNC (SEQ ID NO:60) was confirmed by anti-M13immunostaining on tissue sections (not shown). Strong phage staining wasapparent in tumor vasculature but not in normal vasculature (not shown).Insertless phage did not bind to tumor vessels.

CPRECESIC (SEQ ID NO:56) is a Specific Inhibitor of APA Activity.

To investigate the effect of CPRECESIC (SEQ ID NO:56) on APA enzymeactivity, SK-RC-49/APA cells were incubated with the APA specificsubstrate α-glutamyl-p-nitroanilide in the presence of increasingconcentrations of either CPRECESIC (SEQ ID NO:56) or control GACVRLSACGA(SEQ ID NO:57) peptides. Enzyme activity was evaluated by a colorimetricassay after 2 h incubation at 37° C. CPRECESIC (SEQ ID NO:56) inhibitedAPA enzyme activity, reducing the activity by 60% at the highestconcentration tested. (FIG. 32). The IC₅₀ of CPRECESIC (SEQ ID NO:56)for enzyme inhibition was calculated to be 800 μM. CPRECESIC (SEQ IDNO:56) did not affect the activity of a closely related protease,aminopeptidase N (data not shown).

CPRECESIC (SEQ ID NO:56) Inhibits Migration and Proliferation ofEndothelial Cells.

The potential use of CPRECESIC (SEQ ID NO:56) peptide as ananti-angiogenic drug was determined. First, the effect of APA inhibitionby CPRECESIC (SEQ ID NO:56) peptide in vitro on the migration andproliferation of human umbilical vein endothelial cells (HUVECs)stimulated with VEGF-A (10 ng/ml) was examined. The presence offunctional APA on HUVECs was evaluated by enzyme assay (not shown). Atthe highest concentration tested (1 mM), CPRECESIC (SEQ ID NO:56)peptide inhibited chemotaxis of HUVECs by 70% in a Boyden chamber assay(FIG. 33). At the same peptide concentration, cell proliferation wasinhibited by 50% (FIG. 34). Lower concentrations of CPRECESIC (SEQ IDNO:56) peptide or the GACVRLSACGA (SEQ ID NO:57) control peptide had nosignificant effect on cell migration or proliferation (not shown).

CPRECESIC (SEQ ID NO:56) Inhibits Angiogenesis In Vitro and In Vivo

The inhibitory effect of CPRECESIC (SEQ ID NO:56) peptide in differentin vitro and in vivo models of angiogenesis was examined. HUVECs platedon a three-dimensional matrix gel differentiate into a capillary-likestructure, providing an in vitro model for angiogenesis. Increasingconcentrations of CPRECESIC (SEQ ID NO:56) peptide resulted in aprogressive impairment of the formation of this network (not shown). Ata peptide concentration of 1 mM, vessel-like branching structures weresignificantly fewer and shorter, and as a result, the cells could notform a complete network organization (not shown). The control peptideGACVRLSACGA (SEQ ID NO:57) did not affect HUVEC morphogenesis (notshown).

A commonly used model of simplified in vivo angiogenesis is the chickenchorioallantoic membrane (CAM), in which neovascularization can bestimulated during embryonic development. An appropriate stimulus,adsorbed on a gelatin sponge, induces microvessel recruitment to thesponge itself, accompanied by remodeling and ramification of the newcapillaries. Eight-day-old chicken egg CAMs were stimulated with VEGF-Aalone (20 ng) or with VEGF-A plus CPRECESIC (SEQ ID NO:56) orGACVRLSACGA (SEQ ID NO:57) (1 mM) peptides. The CAMs were photographedat day 12. Neovascularization induced by VEGF-A was inhibited byCPRECESIC (SEQ ID NO:56) by 40% based on the number of capillariesemerging from the sponge (Table 8). The neovessels did not show thehighly branching capillary structures typically seen after VEGF-Astimulation (not shown). Treatment with control peptide GACVRLSACGA (SEQID NO:57) or with lower peptide concentrations of CPRECESIC (SEQ IDNO:56) had no effect on the number of growing vessels (not shown).

TABLE 8 CAM assay for angiogenesis BLOOD TREATMENT VESSEL NUMBERS NoVEGF-A 12.0 ± 2.82* VEGF-A 57.0 ± 1.41* VEGF-A + control 56.5 ± 2.12VEGF-A + CPRECESIC (SEQ ID NO:56)  5.5 ± 1.41* *p <0.01 with theStudent-Newman-Keuls test. The results are expressed as the mean andstandard error from two independent experiments.

APA-Deficient Mice Show Impaired Neovascularization

The ability of APA^(+/−) and APA^(−/−) null mice to undergoneovascularization was examined in a model of hypoxic retinopathy inpremature mice. Induction of retinal neovascularization by relativehypoxia was already present in APA^(+/−) mice compared to wild type mice(not shown). Neovascularization was almost undetectable in APA null mice(not shown). Neovascularization was quantified by counting vitreousprotruding neovascular nuclei from 20 sections of hypoxic eyes.Significant induction of retinal neovascularization (16.17±1.19neovascular nuclei/eye section) was seen in the wild type mice onpostnatal day 17 (P17) after 75% oxygen treatment from P7 to P12.Decreased amounts of neovascular nuclei were seen in the retinas ofAPA^(+/−) (10.76±1.03 neovascular nuclei/eye section) and APA null(4.25±0.45 neovascular nuclei/eye section) mice on P17 after exposure to75% oxygen from P7 to P12.

Discussion

In vivo, APA is overexpressed by activated microvessels, including thosein tumors, but it is barely detectable in quiescent vasculature, makingit a suitable target for vessel-directed tumor therapy. The presentexample identified a novel targeting peptide ligand for APA, CPRECESIC(SEQ ID NO:56). Soluble CPRECESIC (SEQ ID NO:56) peptide inhibited APAenzyme activity with an IC₅₀ of 800 μM.

Using cultured HUVECs as an in vitro model of angiogenesis, solubleCPRECESIC (SEQ ID NO:56) peptide inhibited VEGF-A-induced migration andproliferation of HUVECs. These data are consistent with a requirementfor migration and proliferation of endothelial cells duringangiogenesis. CPRECESIC (SEQ ID NO:56) also blocked the formation ofcapillary-like structures in a Matrigel model and inhibited angiogenesisin VEGF-A-stimulated CAMs.

APA was shown to be an important player in neovascularization induced byrelative hypoxia, since APA null mice had significantly less retinalneovascularization compared to wt mice. These results strengthen thepotential of using APA as a specific target for the inhibition of tumorangiogenesis.

In summary, the soluble peptide CPRECESIC (SEQ ID NO:56) is a selectiveAPA ligand and inhibitor. The inhibition of APA by CPRECESIC (SEQ IDNO:56) led to the inhibition of angiogenesis in different in vitro andin vivo assays, demonstrating for the first time a prominent role forAPA in the angiogenic process. Furthermore, APA-binding phage can hometo tumor blood vessels, suggesting possible therapeutic uses ofCPRECESIC (SEQ ID NO:56) as an inhibitor of tumor neovascularization.The endogenous analog of CPRECESIC (SEQ ID NO:56) may be identified byantibody based purification or identification methods, similar to thosedisclosed.

Example 9 Screening Phage Libraries by PALM

In certain embodiments, it is desirable to be able to select specificcell types from a heterogeneous sample of an organ or tissue. One methodto accomplish such selective sampling is by PALM (Positioning andAblation with Laser Microbeams).

The PALM Robot-Microbeam uses a precise, computer-guided laser formicroablation. A pulsed ultra-violet (UV) laser is interfaced into amicroscope and focused through an objective to a beam spot size of lessthan 1 micrometer in diameter. The principle of laser cutting is alocally restricted ablative photodecomposition process without heating(Hendrix, 1999). The effective laser energy is concentrated on theminute focal spot only and most biological objects are transparent forthe applied laser wavelength. This system appears to be the tool ofchoice for recovery of homogeneous cell populations or even single cellsor subcellular structures for subsequent phage recovery. Tissue samplesmay be retrieved by circumcising a selected zone or a single cell afterphage administration to the subject. A clear-cut gap between selectedand non-selected area is typically obtained. The isolated tissuespecimen can be ejected from the object plane and catapulted directlyinto the cap of a common micro centrifuge tube in an entirelynon-contact manner. The basics of this so called Laser PressureCatapulting (LPC) method is believed to be the laser pressure force thatdevelops under the specimen, caused by the extremely high photon densityof the precisely focused laser microbeam. This tissue harvestingtechnique allows the phage to survive the microdissection procedure andbe rescued.

PALM was used in the present example to select targeting phage for mousepancreatic tissue, as described below.

Materials and Methods

In Vivo and In Situ Panning

A CX₇C peptide phage library (10⁹ TU) was injected into the tail vein ofa C57BL/6 male mouse, and the pancreas was harvested to recover thephage by bacterial infection. Phage from 246 colonies were grownseparately in 5 ml LB/kanamycin (100 μt/ml)/tetracycline (40 μg/ml) at37° C. in the dark with agitation. Overnight cultures were pooled andthe phage purified by NaCl/PEG precipitation for another round of invivo bio-panning. Three hundred colonies were picked from the secondround of panning, and the phage were recovered by precipitation. Phagefrom the second bio-panning round was then used for another round of invivo panning and also was incubated with thawed frozen murine pancreaticsections for one in situ panning round.

For the third in vivo panning round, 10⁹ TU phage from the second roundwere injected into a third mouse and allowed to circulate for sixminutes, followed by an intravenous injection of 50 μl of FITC-lectin(Vector Laboratories, Inc.). After a two-minute circulation, the mousewas perfused through the left ventricle with 3 ml MEM Earle salts. Thepancreas was harvested, frozen at −80° C. in Tissue Tek (Sakura), andsectioned onto prepared slides.

For the third in situ round, purified phage, isolated from the secondround, were incubated with 4-14 μm thawed murine pancreatic sections onice for 30 minutes. Sections were rinsed with 100 μl ice-cold PBS 8× atroom temperature (RT). Bound phage were recovered from each section byadding 100 μl K91 Kan^(R) (OD₆₀₀=2.03) to infect at RT for 30-60minutes. Infected K91 KanR were withdrawn from each section and allowedto recover in 10 ml LB/Kan/Tet (0.2 μg/ml) for 20 minutes in the dark.Aliquots from the each culture were plated out onto LB/Kan/Tet (40μg/ml) plates and incubated overnight in the dark at 37° C. Thetetracycline concentration of the remainder of each culture wasincreased to 40 μg/ml and the cultures were incubated overnight at 37°C. in the dark with agitation for phage amplification and purification.

DNA Amplification

Phage were recovered from cryo-preserved FITC-lectin stained mousepancreatic islets and surrounding acinar cells that were microdissectedfrom 14 μm sections using the PALM (Positioning and Ablation with LaserMicrobeams) cold laser pressure catapulting system. Pancreatic islet andcontrol sections were catapulted into 1 mM EDTA, pH 8, and frozen at−20° C. until enough material was collected for PCR amplification. PhageDNA was amplified with fuSE5 primers: forward primer 5′ TAA TAC GAC TCACTA TAG GGC. AAG CTG ATA AAC CGA TAC AATT 3′ (SEQ ID NO:65), reverseprimer 5′ CCC TCA. TAG TTA GCG TAA CGA TCT 3′ (SEQ ID NO:66). The PCRproducts were subjected to another round of PCR using a nested set ofprimers. The 3′ end of the second primer set was tailed with the M13reverse primer for sequencing purposes. The nested primer set used was:forward nested primer 5′ CCTTTCTATTCTCACTCGGCCG 3′ (SEQ ID NO:67),reverse nested primer 5′ CAGGAAACAGCTATGACCGCTAAACAACTITCAACAGTITCGGC 3′(SEQ ID NO:68). To generate peptide insert sequence containing flankingSfiI restriction sites, two more primers were used: forward libraryprimer 5′CACTCGGCCGACGGGGC 3′ (SEQ ID NO:69), reverse primer5′CAGTTTCGGCCCCAGCGGCCC 3′ (SEQ ID NO:70). PCR products generated fromthe nested primers were gel purified (Qiagen), and confirmed for thepresence of a CX₇C peptide insert sequence using the M13 reverse primerby automated sequencing. PCR products generated from the library primerswere gel purified (Qiagen), ligated into CsCl₂ purified fUSE5/SfiI,electroporated into electrocompetent MC1061 cells, and plated ontoLB/streptomycin (100 μg/ml)/tetracycline (40 μg/ml) agar plates. Singlecolonies were subjected to colony PCR using the fuSE5 primers to verifythe presence of a CX₇C insert sequence by gel electrophoresis. Positiveclones were sequenced using BigDye terminators (Perkin Elmer)

Phage Infection

Pancreatic islet and control sections were catapulted into 1 mM AEBSF,20 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mM elastase inhibitor I, 0.1mM TPCK, 1 nM pepstatin A in PBS, pH 7.4, and frozen for 48 hours orless until enough material was collected. The sections were thawed onice and the volume adjusted to 200 μl with PBS, pH 7.4. Samples wereincubated with 1 ml K91 Kan^(R) (OD=0.22) for two hours at RT on anutator. Each culture was transferred to 1.2 ml LB/Kan/Tet (0.2 μg/ml)and incubated in the dark at RT for 40 minutes. The tetracyclineconcentration was increased to 40 μg/ml for each culture, and thecultures were incubated overnight at 37° C. with agitation. Each culturewas plated out the following day onto LB/Kan/Tet agar plates andincubated for 14 hours at 37° C. in the dark. Positive clones werepicked for colony PCR and automated sequencing.

Results

The general scheme for in vivo panning using PALM is illustrated in FIG.35. After an initial round of in vivo selection, phage were either bulkamplified or else single colonies of phage from pancreas, kidney, lungand adrenal glands were amplified and subjected to additional rounds ofin vivo screening. Both bulk amplified and colony amplified phage frommouse pancreas showed successive enrichment with increasing rounds ofselection (not shown). After three rounds of selection, the colonyamplified phage showed almost an order of magnitude higher enrichmentthan bulk amplified phage (not shown).

Table 9 lists selected targeting sequences and consensus motifsidentified by pancreatic screening.

TABLE 9 Pancreatic targeting peptides and motifs Motif Peptide SequenceGGL CVPGLGGLC (SEQ ID NO:71) (SEQ ID NO:111) CGGLDVRMC (SEQ ID NO:72)CDGGLDWVC (SEQ ID NO:73) LGG CVPGLGGLC (SEQ ID NO:71) (SEQ ID NO:112)CTWLGGREC (SEQ ID NO:74) CSRWGLGGC (SEQ ID NO:75) CPPLGGSRC (SEQ IDNO:48) VRG CVGGVRGGC (SEQ ID NO:76) (SEQ ID NO:113) CVGNDVRGC (SEQ IDNO:77) CESRLVRGC (SEQ ID NO:78) CGGRPVRGC (SEQ ID NO:79) AGG CTPFIAGGC(SEQ ID NO:80) (SEQ ID NO:114) CREWMAGGC (SEQ ID NO:81) CAGGSLRVC (SEQID NO:82) VVG CEGVVGIVC (SEQ ID NO:83) (SEQ ID NO:115) CDSVVGAWC (SEQ IDNO:84) CRTAVVGSC (SEQ ID NO:85) VGG CVGGARALC (SEQ ID NO:86) (SEQ IDNO:116) CVGGVRGGC (SEQ ID NO:76) CLAHRVGGC (SEQ ID NO:88) GGL CWALSGGLC(SEQ ID NO:89) (SEQ ID NO:117) CGGLVAYGC (SEQ ID NO:90) CGGLATTTC (SEQID NO:91) GRY CGRVNSVAC (SEQ ID NO:92) (SEQ ID NO:118) CAGRVALRC (SEQ IDNO:93) GGA CWNGGARAC (SEQ ID NO:94) (SEQ ID NO:119) CLDRGGAHC (SEQ IDNO:95) GVV CELRGVVVC (SEQ ID NO:96) (SEQ ID NO:120) GGV CIGGVHYAC (SEQID NO:97) (SEQ ID NO:121) CGGVHALRC (SEQ ID NO:98) GMWG CIREGMWGC (SEQID NO:99) (SEQ ID NO:122) CIRKGMWGC (SEQ ID NO:100) ALR CGGVHALRC (SEQID NO:98) (SEQ ID NO:123) CAGRVALRC (SEQ ID NO:93) CEALRLRAC (SEQ IDNO:101) ALV CALVNVHLC (SEQ ID NO:102) (SEQ ID NO:124) CALVMVGAC (SEQ IDNO:103) GGVH CGGVHALRC (SEQ ID NO:98) (SEQ ID NO:125) CIGGVHYAC (SEQ IDNO:97) VSG CMVSGVLLC (SEQ ID NO:104) (SEQ ID NO:126) CGLVSGPWC (SEQ IDNO:105) CLYDVSGGC (SEQ ID NO:106) GPW CSKVGPWWC (SEQ ID NO:107) (SEQ IDNO:127) CGLVSGPWC (SEQ ID NO:108) none CAHHALMEC (SEQ ID NO:109)CERPPFLDC (SEQ ID NO:110)

FIG. 36 shows a general protocol for recovery of phage insert sequencesfrom PALM selected thin section materials. As indicated, phage may berecovered by direct infection of E. coli host bacteria, after: proteasedigestion of the thin section sample. Alternatively, phage inserts maybe recovered by PCR amplification and cloned into new vector DNA, thenelectroporated or otherwise transformed into host bacteria for cloning.

Both methods of PALM recovery of phage were successful in retrievingpancreatic targeting sequences. Pancreatic sequences recovered by directbacterial infection included CVPRRWDVC (SEQ ID NO:128), CQHTSGRGC (SEQID NO:129), CRARGWLLC (SEQ ID NO:130), CVSNPRWKC (SEQ ID NO:131),CGGVHALRC (SEQ ID NO:98), CFNRTWIGC (SEQ ID NO:132) and CSRGPAWGC (SEQID NO:133). Pancreatic targeting sequences recovered by amplification ofphage inserts and cloning into phage include CWSRGQGGC (SEQ ID NO:134),CHVLWSIRC (SEQ ID NO:135), CLGLLMAGC (SEQ ID NO:136), CMSSPGVAC (SEQ IDNO:137), CLASGMDAC (SEQ ID NO:138), CHDERTGRC (SEQ ID NO:139), CAHHALMEC(SEQ ID NO:140), CMQGAATSC (SEQ ID NO:141), CMQGARTSC (SEQ ID NO:142)and CVRDLLTGC (SEQ ID NO:143).

FIG. 37 through FIG. 40 show sequence homologies identified for selectedpancreatic targeting sequences. Several proteins known to be present inpancreatic tissues are identified. The results of this example show thatthe PALM method may be used for selecting cell types from tissue thinsections and recovering targeting phage sequences. The skilled artisanwill realize that this method could be used with virtually any tissue toobtain targeting sequences directed to specific types of cells inheterologous organs or tissues.

All of the COMPOSITIONS, METHODS and APPARATUS disclosed and claimedherein can be made and executed without undue experimentation in lightof the present disclosure. While the compositions and methods of thisinvention have been described in terms of preferred embodiments, it areapparent to those of skill in the art that variations maybe applied tothe COMPOSITIONS, METHODS and APPARATUS and in the steps or in thesequence of steps of the methods described herein without departing fromthe concept, spirit and scope of the invention. More specifically, itare apparent that certain agents that are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1.-45. (canceled)
 46. A method of targeting delivery to an adiposetissue, comprising: a) obtaining a peptide comprising the contiguousamino acids of a sequence selected from SEQ ID NO:13, SEQ ID NO:14, SEQID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ IDNO:20, SEQ ID NO:21 or SEQ ID NO:22; and b) administering the peptide toa subject.
 47. The method of claim 46, wherein the subject is a human, amouse, a dog, a cat, a rat, a sheep, a horse, a cow, a goat or a pig.48. The method of claim 46, wherein the targeting peptide has the aminoacid sequence of SEQ ID NO:19 or SEQ ID NO:22
 49. The method of claim46, wherein the peptide is combined with an agent to be delivered to thetissue.
 50. The method of claim 49, wherein the agent is achemotherapeutic agent, a radioisotope, a pro-apoptosis agent, ananti-angiogenic agent, an enzyme, a hormone, a cytokine, a growthfactor, a cytotoxic agent, a peptide, a protein, an antibiotic, anantibody, a Fab fragment of an antibody, an imaging agent, an antigen, asurvival factor, an anti-apoptotic agent, a hormone antagonist, a virus,a bacteriophage, a bacterium, a liposome, a microparticle, a magneticbead, a microdevice, a yeast cell, a mammalian cell, a cell or anexpression vector.
 51. The method of claim 50, where the agent is apro-apoptosis agent selected from the group consisting of gramicidin,magainin, mellitin, defensin, cecropin, (KLAKLAK)₂ (SEQ ID NO:1),(KLAKKLA)₂ (SEQ ID NO:2), (KAAKKAA)₂ (SEQ ID NO:3) and (KLGKKLG)₃ (SEQID NO:4).
 52. The method of claim 51, wherein the pro-apoptosis agent is(KLAKLAK)₂ (SEQ ID NO:1).
 53. The method of claim 49, wherein the agentis an anti-angiogenic agent selected from the group consisting ofthrombospondin, angiostatin5, pigment epithelium-derived factor,angiotensin, laminin peptides, fibronectin peptides, plasminogenactivator inhibitors, tissue metalloproteinase inhibitors, interferons,interleukin 12, platelet factor 4, IF′-10, Gro-B, thrombospondin,2-methoxyoestradiol, proliferin-related protein, carboxiamidotriazole,CM101, Marimastat, pentosan polysulphate, angiopoietin 2 (Regeneron),interferon-alpha, herbimycin A, PNU145156E, 16K prolactin fragment,Linomide, thalidomide, pentoxifylline, genistein, TNP-470, endostatin,paclitaxel, Docetaxel, polyamines, a proteasome inhibitor, a kinaseinhibitor, a signaling peptide, accutin, cidofovir, vincristine,bleomycin, AGM-1470, platelet factor 4 and minocycline.
 54. The methodof claim 49, wherein the agent is a cytokine selected from the groupconsisting of interleukin 1 (IL-1), IL-2, IL-5, IL-10, IL-11, IL-12,IL-18, interferon-γ (IF-γ), IF-α, IF-β, tumor necrosis factor-α (TNF-α),or GM-CSF (granulocyte macrophage colony stimulating factor).
 55. Themethod of claim 49, wherein the agent is a molecule complex that isvirus; a bacteriophage, a bacterium, a liposome, a microparticle, amagnetic bead, a yeast cell, or a cell.
 56. The method of claim 55,wherein the molecular complex is a eukaryotic expression vector.
 57. Themethod of claim 56, wherein said vector is a gene therapy vector. 58.The method of claim 55, wherein said molecular complex is a solidsupport.
 59. The method of claim 49, wherein the agent is a diagnosticagent.
 60. The method of claim 59, wherein the diagnostic agent is animaging agent.
 61. The method of claim 60, wherein the imaging agent,wherein the imaging agent comprises chromium (III), manganese (II), iron(III), iron (II), cobalt (II), nickel (II), copper (II), neodymium(III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II),terbium (III), dysprosium (III), holmium (III) erbium (III), lanthanum(III), gold (III), lead (II), or bismuth (III).
 62. The method of claim60, wherein the agent comprises a radioisotope, and the radioisotope isastatine²¹¹, ¹⁴carbon, ⁵¹chromium, ³⁶chlorine, ⁵⁷cobalt, ⁵⁸cobalt,copper⁶⁷, ¹⁵²Eu, gallium⁶⁷, ³hydrogen, iodine.sup.¹²³, iodine¹²⁵,iodine¹³¹, indium¹¹¹, ⁵⁹iron, ³²phosphorus, rhenium¹⁸⁶, rhenium¹⁸⁸,⁷⁵selenium, ³⁵sulphur, technicium ^(99m) or yttrium⁹⁰.